Pepsin-sensitive modified Bacillus thuringiensis insecticidal toxin

ABSTRACT

The invention relates to the degradation of  Bacillus thuringiensis  Cry proteins in the digestive tracts of mammals and concerns  Bacillus thuringiensis  Cry proteins having a peptide sequence that has been modified in such a way as to make said proteins sensitive to the specific enzymes in the digestive tracts of mammals, in particular pepsins. According to the invention, the Cry proteins are modified by inserting pepsin cleavage sites in the peptide sequence thereof. The invention also relates to transformed plants expressing said modified Cry proteins.

FIELD OF THE INVENTION

[0001] The present invention relates to the degradation of Bacillusthuringiensis Cry proteins in the mammalian digestive tract. It relatesto Bacillus thuringiensis Cry proteins, the peptide sequence of whichhas been modified so as to make them sensitive to the specific enzymesin the mammalian digestive tract, in particular to pepsins. According tothis invention, the Cry proteins are modified by insertion ofpepsin-cleavage sites into their peptide sequence. The invention alsorelates to transformed plants expressing these modified Cry proteins.

[0002] Bacteria of the species Bacillus thuringiensis (hereinafterreferred to as Bt) are well known for the insecticidal toxins which theyproduce. These Gram-positive bacteria form a parasporal crystal proteinduring their stationary phase, which is greatly responsible for theirinsecticidal activity. The crystal protein of Bt consists of aninsecticidal toxin which is protein in nature, referred to as Cryprotein, and encoded by a cry gene. By virtue of its insecticidalproperties, this Cry protein was used in protecting crops against pestinsects, as an alternative solution to synthetic insecticides.Currently, this agronomic use is essentially implemented by two methods,direct spreading of the product as a biopesticide, and genetictransformation of the plants grown, with a gene encoding a Cry protein.Depending on the strains of Bt from which they are derived, the Cryproteins have insecticidal activities with respect to different insectspectra. The main orders of insects against which the Cry toxins areactive are Lepidoptera, Coleoptera and Diptera, but some toxins areeffective with respect to other insect orders. All the Cry proteinsisolated from the various strains of Bt are grouped together in aclassification as a function of their sequence homologies, and they areassigned a code in order to distinguish them (Crickmore et al., 1998,Microbiol. Molec. Biol. Review (62(3), 807-813). The advantage of usingthese toxins in agriculture therefore lies in their specificity ofaction with respect to one or more given insect orders, but also intheir lack of toxicity with respect to mammals, to birds, to amphibiansand to reptiles.

[0003] This lack of toxicity with respect to mammals has made itimpossible to develop the culturing of transgenic plants expressing aCry protein, and to use the seeds from these plants for human and animalfoodstuffs. However, although they are non-toxic with respect tomammals, some of these proteins are relatively undegraded in themammalian digestive tract, and this lack of degradation leads to arelatively long persistence of the toxin in the digestive tract of saidmammals. In addition, a lack of persistence of Cry proteins in themammalian digestive tract is one of the criteria taken into account bythe administrative authorities (for example the United StatesEnvironmental Protection Agency—EPA) which grant marketingauthorizations in the food sector for seeds containing these proteins orfor products derived from these seeds.

BACKGROUND OF THE INVENTION

[0004] The present invention makes it possible to overcome the drawbackmentioned above. This invention is based on the principle according towhich the stability of certain Cry proteins in the mammalian digestivetract is thought to be due to a lack of sensitivity of these proteins tothe specific enzymes in said digestive tract, in particular to theproteases. The solution to this problem therefore lies in the artificialintegration of specific sites, specific to the enzymes of the mammaliandigestive tract, into the Cry protein. A subject of the presentinvention is therefore modified Cry proteins sensitive to the specificenzymes in the mammalian digestive tract, in particular the specificproteases in the mammalian stomach, and more particularly the pepsins.Pepsin is a particular enzyme of the protease family, and it is themajor protease present in the mammalian stomach (95% of stomachproteases). It is an aspartic protease which acts at an optimum pH of 2.Pepsin is an enzyme of choice as a source of degradation of Cry proteinssince it is not present in the digestive tube of insects, in particularof the Lepidoptera, in which the pH of the digestive tube is between 10and 11 (Terra, W. B. and C. Ferreira, 1994, Insect digestive enzymes:properties, compartmentalization and function. Comp. Biochem. Physiol.109B: 1-62). This lack of pepsin in insects therefore guarantees thatintroducing pepsin-specific sites into the Cry proteins does not presenta risk of increasing their degradation in the insect digestive tube. Thepresent invention is therefore a solution to the technical problem setout above, namely an increase in the sensitivity of the Cry proteins toenzymes of the mammalian digestive tract, without alteration of theinsecticidal properties of said Cry proteins.

[0005] However, the Cry protein is a very organized protein, theactivated form of which is made up of three domains, and in which thestructure-function relationships are very strong within and between thedomains. This high level of organization of the Cry proteins does notpermit the random insertion of mutations into the protein. Specifically,the insertion of cleavage sites specific to mammalian stomach enzymesmust not alter the insecticidal properties of the toxins.

[0006] The Cry proteins are naturally produced by the bacterium Bacillusthuringiensis in the form of inactive protoxins. The natural method ofaction of these proteins involves solubilization of the crystal proteinin the insect intestine, proteolytic degradation of the releasedprotoxin, attachment of the activated toxin to the receptors in theinsect intestine, and insertion of the toxin into the apical membrane ofthe intestinal cells so as to create ion channels or pores. Theproteolytic degradation of the protoxin in the insect intestine takesplace under the joint action of the alkaline pH and of the serineproteases (essentially trypsin) of the digestive juice (Schnepf et al.,1998).

[0007] The Cry toxins consist of three structural domains, domain I,domain II and domain III. Domain I occupies approximately the N-terminalhalf of the activated toxin. Domains II and III each occupyapproximately a quarter of the activated toxin. Domain III is located atthe C-terminal end of the activated toxin. Each domain of the Cryprotein has its own structure and its own function.

[0008] Domain I consists of seven α-helices, 6 amphiphilic helices and ahydrophobic helix, connected to one another via inter-helix loopsconsisting of a few amino acids. This domain is the transmembranedomain, responsible for the formation of the ion channel or pore(Aronson et al., 1995; Chen et al., 1993; Manoj-Kumar and Aronson, 1999;Masson et al., 1999; Rang et al., 1999; Coux et al., 1999). Theformation of the transmembrane pore by the α-helices of domain I in factinvolves four Cry proteins which form a complete pore with their fourrespective α4-helices (Masson et al., 1999). A cylindrical pore of fourα4-helices therefore forms. The inside of this pore consists of thehydrophilic faces of the amphiphilic helices; since the negativelycharged residues are present on the hydrophilic faces, they are in thelumen of the pore, in aqueous medium, and perform their ion transportfunction. The outside of the pore consists of the hydrophobic faceswhich anchor the pore in the lipid membrane. The formation of the poreby the α-helices of domain I therefore involves very strongstructure-function relationships and conformational changes over time.The introduction of mutations into the α-helices of domain I thereforehas a high probability of disturbing the function of this domain andtherefore the activity of the toxin.

[0009] Domains II and III of the activated toxin consist of β-sheets,which are themselves also in a very compacted form. These two domainsare involved in receptor site recognition (specificity) and in toxinstability (Abdul-Rauf and Ellar, 1999; Dean et al., 1996; Hussain etal., 1996; Lee et al., 1999; Rajamohan et al., 1996, 1998; Wu and Dean,1996). Domain III exchanges induce changes in specificity (de Maagd etal., 1999). This region is much less conserved, and therefore morevariable, than domain I. It is involved in the specificity of eachtoxin. This variability and these interactions specific to each toxinare involved in the nature of the very specific host spectrum of eachtoxin and are involved in the recognition of different receptor sites.Recognition of the receptor takes place via loops in domain II and indomain III and the conformation of these loops varies subtly from onetoxin to the other as a function of the arrangement and of theinteractions between domains II and III. Domain I also interferes withthe other two domains and influences the general conformation (Rang etal., 1999, 2001). In addition, very little is known about thestructure-function relationships within these two domains, and noinformation is actually available regarding the conformation requiredfor recognition of a receptor site. It is therefore very difficult topredict consequences of introducing modifications into domains II andIII on the specificity, the ability to recognize the receptor sites andthe toxicity of the Cry proteins. Moreover, it is known that mutationsgenerated in domains II and III very often induce destabilization of thetoxin in insects, leading to a loss of toxicity.

[0010] Salt bridges also exist between domains I and II of the Cryproteins. These bridges play an important role in the stability of thetoxin and in the functioning thereof. Artificial elimination of thesebridges in Cry1Aa1 shows that the protoxins and activated toxins areless stable than the parental protein (Vachon et al., 2000). These saltbridges are present between domain II and the 7-helix of domain I. Theacknowledged importance of these bridges implies that mutations indomain II and the α7-helix of domain I have a high risk of disturbingthe function of the Cry proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a pepsin-sensitive modified Cryprotein, characterized in that it has at least one additional pepsincleavage site.

[0012] The term “Cry protein” is intended to mean the insecticidalprotein produced by a strain of bacterium Bacillus thuringiensis(hereinafter referred to as Bt), the various holotypes of which, whichexist and which are to come, are referenced by the Bt classificationcommittee (Crickmore, 2001) and accessible on the Internet site at“www.biols.susx.ac.uk/Home/NeilCrickmore/Bt/index.html.” In particular,this Cry protein is encoded by a cry gene, either naturally by the Btbacterium, or in a recombinant manner in a host organism transformedwith a cry gene or with a gene comprising at least the coding sequenceof a Cry protein. The Cry proteins according to the invention alsocomprise Cry proteins the sequence of which has been artificiallymodified so as to increase their insecticidal activity or theirresistance to treatment conditions. This definition also includes Cryprotein fragments which conserve the insecticidal activity, such as thetruncated Cry proteins comprising only the N-terminal portion of acomplete Cry protein, in particular domain I of this protein (WO94/05771). Also included are the fused Cry proteins, as described ininternational patent application WO 94/24264. Preferably, the Cryprotein according to the invention is selected from the Cry1, Cry3,Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20 proteins.Preferably, it is the Cry9C protein, and preferably the Cry9Ca1 protein(Lambert et al., Appl. Environm. Microbiol. 62, 80-86; WO 94/05771). Inparticular, the present invention also fits any Cry protein, thetoxicity of which has been improved, such as, for example, thosedescribed in patent applications WO 97/49814 or WO 99/00407.

[0013] According to the present invention, the Cry protein is modified.The term “modified Cry protein” is intended to mean a Cry protein, thepeptide sequence of which is different from the sequence of the nativeCry protein from which it is derived. This sequence difference is theresult of artificial modifications introduced by genetic engineering, inparticular the insertion or the substitution of specific amino acidresidues into or in said peptide sequence. In particular, the modifiedCry protein is produced by modification of the nucleotide sequenceencoding it, in particular by the technique of site-directed mutagenesiswell known to those skilled in the art (Hutchinson C. A. et al., 1978,J. Biol. Chem. 253: 6551). Preferably, the modification of the Cryprotein consists of an amino acid residue substitution.

[0014] The modified Cry protein according to the invention ispepsin-sensitive. The pepsin focuses its proteolytic action on specificcleavage sites consisting of the amino acids leucine, phenylalanine andglutamic acid. The proteolysis takes place on the C-terminal side of theresidue concerned. According to the invention, the term“pepsin-sensitive” is intended to mean the property, for the modifiedCry protein, of undergoing proteolysis by pepsin. Proteolysis of the Cryprotein leads to partial or total loss of the insecticidal activity ofsaid protein. The pepsin-sensitivity can therefore be measured bybringing a modified Cry protein according to the invention into contact,preferably in vitro, with a pepsin, and then measuring the loss ofinsecticidal activity of said modified Cry protein in comparison with anative Cry protein which has not been modified according to theinvention. By way of example, the tests described in Examples 7 and 8can be used to measure the pepsin sensitivity of a Cry protein accordingto the invention. Alternatively, the Western blotting technique can alsobe used to measure said pepsin sensitivity. Using this technique, thesensitivity is measured by observing the structural degradation of themodified Cry protein after contact with a pepsin. This observationconsists of the disappearance or the decrease in intensity of a bandcorresponding to the Cry protein on a gel electrophoresis transfermembrane, compared to a native Cry protein which has not been modifiedaccording to the invention. The use of these techniques is part of thegeneral knowledge of those skilled in the art.

[0015] The modified Cry protein according to the invention ischaracterized in that it has at least one additional pepsin cleavagesite. The term “pepsin cleavage site” is intended to mean a siteconsisting of at least one amino acid residue recognized as a site ofproteolysis by pepsin. The amino acid residues recognized by pepsin areleucine, phenylalanine or glutamic acid. The expression “additionalpepsin cleavage site” is intended to mean an additional cleavage sitecompared to the native Cry protein as produced by the Bt bacterium.

[0016] Preferably, the additional pepsin cleavage site is represented byan amino acid residue selected from leucine, phenylalanine or glutamicacid residues. According to a particular embodiment of the invention,the modified Cry protein has several additional pepsin cleavage sitesrepresented by the same amino acid residue. According to anotherembodiment of the invention, the modified Cry protein has severaladditional pepsin cleavage sites represented by different amino acidresidues.

[0017] According to a particular embodiment of the invention, themodified Cry protein according to the invention is characterized in thatit has at least one additional pepsin cleavage site in at least one ofthe inter-α-helix loops of domain I. The expression “inter-α-helix loopsof domain I” is intended to mean the peptide chains linking the sevenα-helices of domain I of the Cry proteins as described in Grochulski etal. (1995) and Li et al. (1991). According to the invention, the Cryprotein should have at least one additional pepsin cleavage site. Inaddition, said additional cleavage site is in at least one of theinter-α-helix loops of domain I. The term “additional” is thereforeunderstood to be supplementary compared to the number of pepsin cleavagesites naturally present in the inter-α-helix loops of domain I of thenative Cry protein as produced by the Bt bacterium. This definitionmeans that the modified Cry protein according to the invention ischaracterized in that it has a number of pepsin cleavage sites in itsinter-α-helix loops of domain I which is greater than the number ofthese sites in the same native Cry protein as produced by the Btbacterium, the difference between said numbers being at least equal to1.

[0018] According to a particular embodiment of the invention, themodified Cry protein according to the invention has at least one pepsincleavage site in the inter-α-helix loop linking the α3 and α4 helices ofdomain I.

[0019] According to a preferred embodiment of the invention, themodified Cry protein is a modified Cry9C protein. Preferably, themodified Cry protein is a modified Cry9Ca1 protein having a pepsincleavage site positioned on amino acid residue 164. In particular, thearginine residue naturally present at position 164 on the Cry9Ca1protein is replaced with an amino acid residue chosen from leucine,phenylalanine and glutamic acid residues, on the Cry9Ca1 proteinmodified according to the invention. Preferably, the Cry9Ca1 proteinmodified according to the invention is selected from the Cry proteinsthe sequences of which are represented by the identifiers SEQ ID NO:4,SEQ ID NO:6 or SEQ ID NO:8.

[0020] The present invention also relates to a pepsin-sensitive modifiedCry protein, characterized in that the additional pepsin cleavage siteswhich it possesses are introduced by substituting aspartic acid residueswith glutamic acid residues, substituting tryptophan residues withphenylalanine residues, and substituting valine or isoleucine residueswith leucine residues. Preferably, the degree of substitution which saidmodified Cry protein has is 25%. The expression “degree of substitution”is intended to mean the percentage of amino acid residues of the nativeCry protein which are replaced with amino acid residues corresponding topepsin cleavage sites in the modified Cry protein of the invention.

[0021] A subject of the present invention is also a method forincreasing the pepsin sensitivity of the Cry proteins, characterized inthat at least one additional pepsin cleavage site is introduced intosaid Cry proteins. The expression “increasing the pepsin sensitivity ofthe Cry proteins” is intended to mean an increase in the pepsinsensitivity of the Cry proteins obtained by said method compared to thecorresponding native Cry proteins, this increase resulting inproteolytic destruction and a loss of insecticidal activity of the Cryproteins, these effects possibly being partial or total.

[0022] The introduction of at least one pepsin cleavage site is carriedout artificially by genetic engineering. In particular, it involves aninsertion or a substitution of amino acid residues. Preferably, itinvolves a substitution. Such a substitution can be readily carried outby the site-directed mutagenesis technique well known to those skilledin the art.

[0023] Preferably, the Cry protein to which the method according to theinvention applies is selected from the Cry1, Cry3, Cry4, Cry7, Cry8,Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20 proteins. Preferably, it isthe Cry9C protein, and preferably the Cry9Ca1 protein.

[0024] In particular, the additional pepsin cleavage site is representedby an amino acid residue chosen from leucine, phenylalanine and glutamicacid residues.

[0025] According to a particular embodiment of the invention, the methodaccording to the invention is characterized in that at least oneadditional pepsin cleavage site is introduced into at least one of theinter-α-helix loops of domain I of said Cry protein.

[0026] According to another particular embodiment of the invention, themethod according to the invention is characterized in that at least oneadditional pepsin cleavage site is introduced into the inter-α-helixloop linking the α3 and α4 helices of domain I.

[0027] According to a preferred embodiment of the invention, the presentmethod applies to a Cry9C protein. Preferably, it applies to a Cry9Ca1protein, and the additional pepsin cleavage site is introduced bysubstitution of amino acid residue 164. In particular, the arginineresidue naturally present at position 164 on the Cry9Ca1 protein isreplaced with an amino acid residue chosen from leucine, phenyl-alanineand glutamic acid residues.

[0028] The present invention also relates to a method for increasing thepepsin sensitivity of the Cry proteins, characterized in that theadditional pepsin cleavage sites are introduced by substituting asparticacid residues with glutamic acid residues, substituting tryptophanresidues with phenylalanine residues, and substituting valine orisoleucine residues with leucine residues.

[0029] Preferably, the degree of substitution introduced into said Cryprotein is 25%.

[0030] The present invention also relates to a polynucleotide encoding amodified Cry protein according to the invention. According to thepresent invention, the term “polynucleotide” is intended to mean anatural or artificial nucleotide sequence which may be of the DNA or RNAtype, preferably of the DNA type, in particular double-stranded.

[0031] The present invention also relates to a chimeric gene comprising,functionally linked to one another, at least one promoter which isfunctional in a host organism, a polynucleotide encoding a modified Cryprotein according to the invention, and a terminator element which isfunctional in this same host organism. The various elements which achimeric gene can contain are, firstly, regulatory elements for thetranscription, the translation and the maturation of proteins, such as apromoter, a sequence encoding a signal peptide or a transit peptide, ora terminator element constituting a polyadenylation signal and,secondly, a polynucleotide encoding a protein. The expression“functionally linked to one another” means that said elements of thechimeric gene are linked to one another in such a way that thefunctioning of one of these elements is affected by that of another. Byway of example, a promoter is functionally linked to a coding sequencewhen it is capable of affecting the expression of said coding sequence.The construction of the chimeric gene according to the invention and theassembly of its various elements can be carried out using techniqueswell known to those skilled in the art, in particular those described inSambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Nolan C.ed., New York: Cold Spring Harbor Laboratory Press). The choice of theregulatory elements constituting the chimeric gene depends essentiallyon the host species in which they must function, and those skilled inthe art are capable of selecting regulatory elements which arefunctional in a given host organism. The term “functional” is intendedto mean capable of functioning in a given host organism.

[0032] According to a particular embodiment of the invention, thechimeric gene contains a “constitutive” promoter. A constitutivepromoter according to the present invention is a promoter which inducesthe expression of a coding sequence in all the tissues of a hostorganism and continuously, i.e. during the entire duration of the lifecycle of said host organism. Some of these promoters can betissue-specific, i.e. can express the coding sequence continuously, butonly in a particular tissue of the host organism. Constitutive promoterscan originate from any type of organism. Among the constitutivepromoters which may be used in the chimeric gene of the presentinvention, mention may be made, by way of example, of bacterialpromoters, such as that of the octopine synthase gene or that of thenopaline synthase gene, viral promoters, such as that of the genecontrolling transcription of the 19S or 35S RNAs of the cauliflowermosaic virus (Odell et al., 1985, Nature, 313, 810-812), or thepromoters of the cassava vein mosaic virus (as described in patentapplication WO 97/48819). Among the promoters of plant origin, mentionwill be made of the promoter of the ribulose-biscarboxylase/oxygenase(RuBisCO) small subunit gene, the promoter of a histone gene asdescribed in application EP 0 507 698, the promoter of the EF1-α gene(WO 90/02172), the promoter of an actin gene (U.S. Pat. No. 5,641,876),or the promoter of a ubiquitin gene (EP 0342926).

[0033] According to another particular embodiment of the invention, thechimeric gene contains an inducible promoter. An inducible promoter is apromoter which only functions, i.e. which only induces expression of acoding sequence, when it is itself induced by an inducing agent. Thisinducing agent is generally a substance which can be synthesized in thehost organism subsequent to a stimulus external to said organism, thisexternal stimulus possibly being physical or chemical, biotic or abioticin nature. Such promoters are known, such as, for example, the promoterof the plant O-methyltransferase class II (COMT II) gene described inpatent application WO 00/56897, the Arabidopsis PR-1 promoter (Lebel etal., 1998, Plant J. 16(2): 223-233), the EAS4 promoter of the tobaccosesquiterpene synthase gene (Yin et al., 1997, Plant Physiol. 115(2),437-451), or the promoter of the gene encoding3-hydroxy-3-methylglutaryl coenzyme A reductase (Nelson et al., 1994,Plant Mol. Biol. 25(3): 401-412).

[0034] Among the terminator elements which can be used in the chimericgene of the present invention, mention may, for example, be made of thenos terminator element of the gene encoding Agrobacterium tumefaciensnopaline synthase (Bevan et al., 1983, Nucleic Acids Res. 11(2),369-385), or the terminator element of a histone gene as described inapplication EP 0 633 317.

[0035] According to a particular embodiment of the invention, thepromoter and the terminator element of the chimeric gene according tothe invention are both functional in plants.

[0036] It also appears to be important for the chimeric gene toadditionally comprise a signal peptide or a transit peptide which makesit possible to control and orient the production of the Cry proteinspecifically in a cellular compartment of the host organism, such as,for example, the cytoplasm, in a particular compartment of thecytoplasm, or the cell membrane or, in the case of plants, in aparticular type of cellular compartment, for example the chloroplasts,or in the extracellular matrix.

[0037] The transit peptides can be either single or double. The doubletransit peptides are optionally separated by an intermediate sequence,i.e. they comprise, in the direction of transcription, a sequenceencoding a transit peptide of a plant gene encoding an enzyme located inplastids, a portion of sequence of the mature N-terminal portion of aplant gene encoding an enzyme located in plastids, and then a sequenceencoding a second transit peptide of a plant gene encoding an enzymelocated in plastids. Such double transit peptides are, for example,described in patent application EP 0 508 909.

[0038] Signal peptides of use according to the invention which may bementioned include in particular the signal peptide of the tobacco PR-1αgene described by Cornelissen et al. (1987, Nucleic Acid Res. 15,6799-6811), in particular when the chimeric gene according to theinvention is introduced into plant cells or plants.

[0039] The present invention also relates to a vector containing achimeric gene according to the invention. Such a vector is of use fortransforming a host organism and expressing a modified Cry proteinaccording to the invention in said organism. This vector may be aplasmid, a cosmid, a bacteriophage or a virus. In general, the mainqualities of this vector should be an ability to maintain itself and toself-replicate in the host organism's cells, in particular by virtue ofthe presence of an origin of replication, and to express therein amodified Cry protein. The choice of such a vector and also thetechniques for inserting the chimeric gene according to the inventiontherein are widely described in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Nolan C. ed., New York: Cold Spring HarborLaboratory Press) and are part of the general knowledge of those skilledin the art. The vector used in the present invention may also contain,in addition to the chimeric gene of the invention, a chimeric genecontaining a selectable marker. This selectable marker makes it possibleto select the host organisms effectively transformed, i.e. those havingincorporated the vector. Among the selectable markers which can be usedin many host organisms, mention may be made of markers containing genesfor resistance to antibiotics, such as that of the hygromycinphosphotransferase gene (Gritz et al., 1983, Gene 25: 179-188).Preferably, the host organism to be transformed is a plant. Among theselectable markers which can be used in plants, mention may be made ofmarkers containing genes for tolerance to herbicides, such as the bargene (White et al., NAR 18: 1062, 1990) for tolerance to bialaphos, theEPSPS gene (U.S. Pat. No. 5,188,642) for tolerance to glyphosate or elsethe HPPD gene (WO 96/38567) for tolerance to isoxazoles. Mention mayalso be made of genes encoding readily identifiable enzymes such as theGUS enzyme, or genes encoding pigments or enzymes which regulate theproduction of pigments in the transformed cells. Such selectable markergenes are in particular described in patent applications WO 91/02071 andWO 95/06128.

[0040] The present invention also relates to host organisms transformedwith a vector as described above. The term “host organisms” is intendedto mean any type of organism, in particular plants or microorganismssuch as bacteria, viruses, fungi or yeast. The term “transformed hostorganism” is intended to mean a host organism which has incorporatedinto its genome the chimeric gene of the invention and, consequently,produces a modified Cry protein according to the invention in itstissues. To obtain the host organisms according to the invention, thoseskilled in the art can use one of the many known methods oftransformation. One of these methods consists in bringing the cells tobe transformed into contact with polyethylene glycol (PEG) and thevectors of the invention (Chang and Cohen, 1979, Mol. Gen. Genet.168(1), 111-115; Mercenier and Chassy, 1988, Biochimie 70(4), 503-517).Electroporation is another method, which consists in subjecting thecells or tissues to be transformed and the vectors of the invention toan electric field (Andreason and Evans, 1988, Biotechniques 6(7),650-660; Shigckawa and Dower, 1989, Aust. J. Biotechnol. 3(1), 56-62).Another method consists in directly injecting the vectors into the hostcells or tissues by microinjection (Gordon and Ruddle, 1985, Gene(33(2), 121-136). Advantageously, the “biolistic” method may be used. Itconsists in bombarding cells or tissues with particles onto which thevectors of the invention are adsorbed (Bruce et al., 1989, Proc. Natl.Acad. Sci. US 86(24), 9692-9696; Klein et al., 1992, Biotechnology10(3), 286-291; U.S. Pat. No. 4,945,050). Preferably, the transformationof plants will be carried out using bacteria of the Agrobacterium genus,preferably by infecting the cells or tissues of said plants with A.tumefaciens (Knopf, 1979, Subcell. Biochem. 6, 143-173; Shaw et al.,1983, Gene 23(3): 315-330) or A. rhizogenes (Bevan and Chilton, 1982,Annu. Rev. Genet. 16: 357-384; Tepfer and Casse-Delbart, 1987,Microbiol. Sci. 4(1), 24-28). Preferably, the transformation of plantcells with Agrobacterium tumefaciens is carried out according to theprotocol described by Ishida et al. (1996, Nat. Biotechnol. 14(6),745-750).

[0041] These various techniques are in particular described in thefollowing patents and patent applications: U.S. Pat. Nos. 4,459,355,4,536,475, 5,464,763, 5,177,010, 5,187,073, EP 267,159, EP 604 662, EP672 752, U.S. Pat. Nos. 4,945,050, 5,036,006, 5,100,792, 5,371,014,5,478,744, 5,179,022, 5,565,346, 5,484,956, 5,508,468, 5,538,877,5,554,798, 5,489,520, 5,510,318, 5,204,253, 5,405,765, EP 270 615, EP442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 andWO 95/06128.

[0042] The present invention also relates to a method for producing themodified Cry proteins according to the invention. This method comprisesat least the steps of:

[0043] a) culturing a transformed host organism according to theinvention in a culture medium suitable for the growth and for themultiplication of said organism,

[0044] b) extracting the Cry proteins produced by the transformedorganism cultured in step (a).

[0045] Depending on the host organism chosen to carry out this methodand depending on the chimeric gene which it contains, the Cry proteinsproduced are either produced in the host organism, or are secreted intothe culture medium. It ensues that the extraction provided for in step(b) may require a step for destroying the microorganisms, or at leastthe cells of which they are composed, in order to release the Cryproteins if said proteins are not secreted into the culture medium. Theextraction step common to the two possibilities (proteins secreted ornot secreted) consists of removal of the host organisms or debris fromthese organisms by filtration or centrifugation of the culture medium.

[0046] According to a particular embodiment, this method for producingthe modified Cry proteins can also comprise an additional step (c) ofpurification of the Cry proteins produced, from the culture medium.

[0047] According to a preferred embodiment, the host organism is amicroorganism. Preferably, the host organism is a Bacillus thuringiensisbacterium and the culturing performed in step (a) is continued until thesporulation phase of said bacteria.

[0048] The present invention also comprises plants transformed with avector according to the invention, characterized in that they contain achimeric gene according to the invention stably integrated into theirgenome, and express a modified Cry protein in their tissues. Theinvention also extends to the parts of these plants, and the descendantsof these plants. The expression “part of these plants” is intended tomean any organ of these plants, whether it is aerial or subterranean.The aerial organs are the stems, the leaves and the flowers. Thesubterranean organs are mainly the roots, but they can also be tubers.The term “descendants” is intended to mean mainly the seeds containingthe embryos derived from the reproduction of these plants with oneanother. By extension, the term “descendants” applies to all the plantsand seeds formed in each new generation derived from crosses between aplant, in particular a plant variety, and a transformed plant accordingto the invention.

[0049] The transformed plants according to the invention may bemonocotyledones or dicotyledones. Preferably, these plants are plants ofagronomic value. Advantageously, the monocotyledonous plants are wheat,maize and rice. Advantageously, the dicotyledonous plants are rapeseed,soybean, tobacco and cotton.

[0050] According to a particular embodiment of the invention, thetransformed plants according to the invention contain, in addition to achimeric gene according to the invention, at least one other genecontaining a polynucleotide encoding a protein of interest. Among thepolynucleotides encoding a protein of interest, mention may be made ofpolynucleotides encoding an enzyme for resistance to a herbicide, forexample the polynucleotide encoding the bar enzyme (White et al., NAR18: 1062, 1990) for tolerance to bialaphos, the polynucleotide encodingthe EPSPS enzyme (U.S. Pat. No. 5,188,642; WO 97/04103) for tolerance toglyphosate or else the polynucleotide encoding the HPPD enzyme (WO96/38567) for tolerance to isoxazoles. Also contained in these plantsmay be polynucleotides for resistance to diseases, for example apolynucleotide encoding the oxalate oxidase enzyme as described inpatent application EP 0 531 498 or U.S. Pat. No. 5,866,788, or apolynucleotide encoding an antibacterial and/or antifungal peptide suchas those described in patent applications WO 97/30082, WO 99/24594, WO99/02717, WO 99/53053 and WO 99/91089. Mention may also be made ofpolynucleotides encoding agronomic characteristics of the plant, inparticular a polynucleotide encoding a Δ-6 desaturase enzyme asdescribed in U.S. Pat. Nos. 5,552,306 and 5,614,313, and patentapplications WO 98/46763 and WO 98/46764, or a polynucleotide encoding aserine acetyltransferase (SAT) enzyme as described in patentapplications WO 00/01833 and PCT/FR 99/03179. The transformed plantsaccording to the invention can also contain a polynucleotide encodinganother insecticidal toxin, for example a polynucleotide encodinganother Bacillus thuringiensis Cry protein (for example, seeinternational patent application WO 98/40490).

[0051] A subject of the present invention is also monoclonal orpolyclonal antibodies directed against a modified Cry protein accordingto the invention, or a fragment thereof. The techniques for producingantibodies are widely described in the general literature and inreference works such as Immunological Techniques Made Easy (1998, 0.Cochet, J. -L. Teillaud, C. Sautès eds., John Wiley & Sons, Chichester).Preferably, the antibodies according to the invention are used in tests,or kits, for detecting the Cry proteins according to the invention.

[0052] The examples below make it possible to illustrate the presentinvention without, however, limiting the scope thereof.

EXAMPLES Example 1 Creation of a Pepsin Cleavage Site at Amino Acid 164of the Cry9Ca1 Toxin

[0053] A pepsin-specific site is introduced into the Bacillusthuringiensis Cry9Ca1 toxin by substituting the arginine naturallypresent at position 164 in this toxin with one of the three amino acidsrecognized by pepsin: leucine, phenylalanine or glutamic acid. Aminoacid 164 is present in the inter-α-helix loop linking the α3 and α4helices of domain I (hereinafter referred to as α3-α4 inter-helix loop)

[0054] The native sequence of the α3-α4 inter-helix loop is betweenaspartic acid 159 and valine 168. The sequence of this loop is asfollows: DRNDTRNLSV. This amino acid sequence corresponds to thefollowing DNA sequence extending from base 475 to base 504: GAT CGA AATGAT ACA CGA AAT TTA AGT GTT Asp Arg Asn Asp Thr Arg Asn Leu Ser Val

[0055] Codon 164 (CGA) encoding arginine is modified to a codon encodingeither leucine or phenylalanine or glutamic acid. The codonpossibilities are as follows: Leucine: TTA, TTG, CTT, CTC, CTA or CTGPhenylalanine: TTT or TTC Glutamic acid: GAA or GAG

[0056] The choice of preferential codons in the site-directedmutagenesis depends on the organism in which the modified cry gene mustbe expressed and therefore varies accordingly. This choice is part ofthe general knowledge of those skilled in the art, who will adapt thepreferential codons as a function of the chosen organism for production.In this example, the chosen organism for expression is the B.thuringiensis bacterium. The codons preferentially used by B.thuringiensis to encode leucine, phenylalanine or glutamic acid are,respectively, TTA (leucine), TTT (phenylalanine) and GAA (glutamicacid).

[0057] The modification for expression in Bt can therefore be carriedout using the following mutagenesis oligonucleotides (in theoligonucleotides described in the examples below, the codon in uppercase letters corresponds to the mutated codon, and the bases and aminoacids in bold characters correspond to the bases and amino acidsspecifically mutated): Oligonucleotide No. 1: 5′-gat cga aat gat aca TTAaat tta agt gtt gtt-3′    Asp Arg Asn Asp Thr Leu Asn Leu Ser Val Val

[0058] Oligonucleotide No. 1 allows the replacement of arginine 164 witha leucine. Oligonucleotide No. 2: 5′-gat cga aat gat aca TTT aat tta agtgtt gtt-3′    Asp Arg Asn Asp Thr Phe Asn Leu Ser Val Val

[0059] Oligonucleotide No. 2 allows replacement of arginine 164 with aphenylalanine. Oligonucleotide No. 3: 5′-gat cga aat gat aca GAA aat ttaagt gtt gtt-3′    Asp Arg Asn Asp Thr Glu Asn Leu Ser Val Val

[0060] Oligonucleotide No. 3 allows replacement of arginine 164 with aglutamic acid.

[0061] The characteristics of the bacterial strains of Escherichia coliused to modify the sequence of the cry9Ca1 gene are as follows:

[0062] JM 109 of genotype recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiD(lac-proAb) F′ (traD36 proAB+lacIq lacZ DM15)

[0063] BMH 71-18 mut S of genotype thi, supE, (lac-proAB),(mutS::Tn10)(F′, proAB, lacIqZΔM15).

[0064] The plasmid DNA is prepared by minipreparation according to thealkaline lysis technique (Birboim and Doly, 1979). Each bacterial colonyis grown in 2 ml of LB medium supplemented with the appropriateantibiotic, overnight at 37° C. with shaking (200 rpm). The culture isthen transferred into a microtube and then centrifuged at 13 500 g for 5min. After removal of the supernatant, the bacteria are resuspended in100 μl of a solution of 25 mM Tris-HCl, pH 8, and 10 mM EDTA containingRNase A at a final concentration of 100 μg/ml. 200 μl of a 0.2 M NaOHsolution containing 1% SDS are added and the suspension is mixed twiceby inverting the microtube. 150 μl of a 2.55 M potassium acetatesolution, pH 4.5, are added and the suspension is incubated for 5 min inice. After centrifugation for 15 min at 13 500 g, the supernatant istransferred into a microtube containing 1 ml of cold ethanol. Aftercentrifugation for 30 min at 13 500 g, the supernatant is removed andthe pellet is washed with 1 ml of 70% ethanol. The pellet containing theDNA is dried for a few minutes under vacuum and then taken up in 50 μlof sterile distilled water. The samples are then placed at 65° C. for 30min.

[0065] The digestions with restriction endonucleases are carried out for1 μg of DNA in a final volume of 20 μl in the presence of one tenth ofthe final volume of 10× buffer recommended by the supplier for eachenzyme, and using 5 units of enzyme. The reaction is incubated for 2 to3 h at the optimal temperature for the enzyme.

[0066] Dephosphorylation of the 5′ ends engendered by restriction enzymeis carried out with calf intestine alkaline phosphatase. The reaction iscarried out using 5 μl of 10× dephosphorylation buffer (500 mM Tris-HCl,pH 9.3, 10 mM MgCl2, 1 mM ZnCl₂ and 10 mM spermidine) and one unit ofenzyme per μg of DNA in a final volume of 50 μl. The reaction isincubated for one hour at 37° C. in the case of overhanging 5′ ends orat 55° C. in the case of blunt ends or 3′ overhanging ends. Afterdephosphorylation, the enzyme is then inactivated for 30 min at 65° C.and then removed with two volume for volume extractions with aphenol-chloroform-isoamyl alcohol (25-24-1) mixture. The ligations areformed using T4 phage DNA ligase. They are carried out with an amount ofvector equal to 100 ng and an insert/vector molar ratio of between 5 and10. The final volume of the reaction is 30 μl and comprises 3 μl of 10×ligation buffer (300 mM Tris-HCl, pH 7.8, 100 mM MgCl₂, 100 mM DTT and10 mM ATP) and 3 units of enzyme. The reaction is incubated overnight at14° C.

[0067] The mutagenesis oligonucleotide (oligonucleotide No. 1,oligonucleotide No. 2 and oligonucleotide No. 3) are phosphorylated inthe 5′ position in order to allow ligation. 100 pmol of oligonucleotideare incubated for 30 min at 37° C. with 5 units of T4 polynucleotidekinase in a final volume of 25 μl in the presence of 2.5 μl of 10×phosphorylation buffer (700 mM TrisHCl, pH 7.6, 100 mM MgC12 and 50 mMDTT) in the presence of ATP at a final concentration of 1 mM. The enzymeis then inactivated at 70° C. for 10 min.

[0068] The site-directed mutagenesis is carried out according to aconventional method described below. Other procedures known to thoseskilled in the art are described in the literature and give identicalresults. The site-directed mutagenesis method used is that described bythe manufacturer for the use of the Altered Sites II system marketed bythe company Promega. A detailed description of the mutagenesis systemand of the protocol can be found on the internet site of the companyPromega at the address http://www.promega.com. The cry9Ca1 gene ispre-cloned into a phagemide pAlter-1 (Promega) carrying the tetracyclineresistance gene and the ampicillin resistance gene containing a pointmutation. The DNA fragment to be mutated is pre-cloned into the plasmidpAlter-1. 0.5 pmol of plasmid DNA are denatured by adding 2 μl of 2 MNaOH, 2 mM EDTA in a final volume of 20 μl and incubating for 5 min atambient temperature. 2 μl of 2 M ammonium acetate, pH 4.6, and 75 μl ofethanol are added and the mixture is incubated at −70° C. for 30 min.After centrifugation at 14 000 g for 15 min at 4° C., the pellet is thenrinsed with 200 μl of 70% ethanol and recentrifuged at 14 000 g for 15min at 4° C. The denatured DNA pellet is then dried under vacuum andresuspended in 100 μl of sterile distilled water. 10 μl of denaturedDNA, i.e. 0.05 pmol, are mixed with 0.25 pmol of phosphorylatedampicillin-resistance gene repair oligonucleotide, 0.25 pmol oftetracycline-resistance gene destruction oligonucleotide and 1.25 pmolof phosphorylated mutagenesis oligonucleotide (oligonucleotide No. 1,No. 2 or No 3) in the presence of hybridization buffer (20 mM Tris-HCl,pH 7.5, 10 mM MgCl2, 50 mM NaCl) and incubated at 75° C. for 5 min, andthen slowly cooled to ambient temperature. 5 μl of sterile distilledwater, 3 μl of 10× synthesis buffer (100 mM Tris-HCl, pH 7.5, 20 mM DTT,10 mM ATP, 5 mM dNTP), 10 units of T4 DNA polymerase and 3 units of T4DNA ligase are added and the reaction is incubated for 90 min at 37° C.200 μl of competent E. coli BMH 71-18 bacteria are then incubated in thepresence of 1.5 μl of the preceding reaction, in ice for 30 min. A heatshock is then performed by placing the bacteria at 42° C. for 50 sec andthen in ice for 2 min. 900 μl of LB medium are then added and thesuspension is incubated at 37° C. for one hour with shaking. 4 ml of LBmedium supplemented with ampicillin at the final concentration of 100μg/ml are then added and the culture is incubated overnight at 37° C.with shaking. A minipreparation of plasmid DNA is prepared from the 4 mlof culture according to the plasmid DNA extraction protocol describedabove. 200 μl of competent E. coli JM109 bacteria are then incubated inthe presence of 1 ng of plasmid DNA, in ice for 30 min. A heat shock isthen performed by placing the bacteria at 42° C. for 50 sec, and then inice for 2 min. 900 μl of LB medium are then added and the suspension isincubated at 37° C. overnight with shaking. 100 μl of bacterialsuspension are then plated out on a Petri dish containing solid LBmedium supplemented with ampicillin at the final concentration of 100μg/ml. The recombinants obtained are screened in order to find the cloneof interest. This search is carried out by isolating the plasmid DNA ofseveral colonies by the minipreparation technique described above, andthen by sequencing this DNA. The recombinants are then selected usingmedium supplemented with tetracycline at the final concentration of 12.5μg/ml. The correctness of the desired mutation and the verification ofthe lack of undesirable mutations are controlled by sequencing the DNAafter site-directed mutagenesis. DNA samples for the sequencing arepurified with the Wizard Plus SV Minipreps DNA Purification System(Promega) according to the procedure recommended by the supplier, andthe sequencing is carried out on an ABI 377 automatic sequencer(Perkin-Elmer) using sequencing reactions carried out according to thechain termination method (Sanger et al., 1977), by PCR using the ABIPRISM BigDye terminator Cycle Sequencing Kit system. For carrying outthe sequencing reactions and the automatic analysis of the samples, theprocedures used are those recommended by the supplier (AppliedBiosystems).

Example 2 Creation of Pepsin Cleavage Sites in the α3-α4 Inter-helixLoop of the Cry9Ca1 Toxin

[0069] Pepsin-specific sites are introduced into the α3-α4 inter-helixloop of the Cry9Ca1 toxin by substituting at least one amino acid ofthis inter-helix loop with an amino acid recognized by pepsin, namelyleucine, phenylalanine and glutamic acid. Codons encoding these threeamino acids will therefore be created in place of the codons naturallypresent in the region extending from base 475 to base 504. The codonpossibilities for these three amino acids are described in Example 1.

[0070] As in Example 1, the selected organism for producing the modifiedCry protein is the B. thuringiensis bacterium, and the choice of thereplacement codons is therefore identical to that of Example 1. Inaddition, if another organism for production is selected, those skilledin the art will be able to adjust the preferential codons as a functionof the organism for production selected.

[0071] Various alternative sequences for the α3-α4 inter-helix loop arepossible, each having a variable number of leucine, phenylalanine orglutamic acid residues. Some of these possibilities are given inTable 1. The possibilities for modification of the α3-α-4 inter-helixloop are not limited to those given in Table 1 below. The aim of thelist given in Table 1 is to illustrate some of the possibilities formodification without limiting the scope of the invention to theseillustrations. Those skilled in the art, being aware of the codonsspecific for each amino acid according to the organisms, will be able toadapt the teaching described in this example to all the possibilitiesfor modifying the α3-α4 inter-helix loop, in particular to those whichare not described in Table 1. TABLE 1 Examples of possible modificationsof the α3-α4 inter-helix loop of the Cry9Ca1 toxin Amino acid Proteinsequence Nucleotide sequence CryCa1 DRNDTRNLSV gat cga aat gat aca cgaaat tta agt gtt Asp Arg Asn Asp Thr Arg Asn Leu Ser Val Mutant No. 1ELNEFLNSV gaA TTA aat gaA TTT TTa aat tta agt gtt Glu Leu Asn Glu PheLeu Asn Leu Ser Val Mutant No. 2 ELNELLNLSV gaA TTA aat gaA TTa TTa aattta agt gtt Glu Leu Asn Glu Leu Leu Asn Leu Ser Val Mutant No. 3ELLEFLLLSV gaA TTa TTA gaA TTT TTa TTA tta agt gtt Glu Leu Leu Glu PheLeu Leu Leu Ser Val Mutant No. 4 ELLELLLLSV gaA TTa TTA gaA TTa TTa TTAtta agt gtt Glu Leu Leu Glu Leu Leu Leu Leu Ser Val Mutant No. 5ELLEELLLSV gaA TTa TTA gaA GAa TTa TTa tta agt gtt Glu Leu Leu Glu GluLeu Leu Leu Ser Val Mutant No. 6 ERLEFLLLSV gaA cga TTA gaA TTT TTa TTAtta agt gtt Glu Arg Leu Glu Phe Leu Leu Leu Ser Val Mutant No. 7ERLELLLLSV gaA cga TTA gaA TTa TTa TTA tta agt gtt Glu Arg Leu Glu LeuLeu Leu Leu Ser Val Mutant No. 8 ERLEELLLSV gaA TTa GAA gaA TTa TTa TTAtta agt gtt Glu Leu Glu Glu Leu Leu Leu Leu Ser Val Mutant No. 9ELLEEEELSV gaA TTa TTA gaA GAa GAa GAA tta agt gtt Glu Leu Leu Glu GluGlu Glu Leu Ser Val

[0072] The substitution of several amino acids within the α3-α4inter-helix loop requires, for each of the mutants, the successive useof several mutagenesis oligonucleotides. The mutagenesisoligonucleotides required to create the examples of mutants given inTable 1 are presented below (numbered from 4 to 20). Oligonucleotide No.4: cga aat gat aca cga TTA tta agt gtt gtt cgt Arg Asn Asp Thr Arg LeuLeu Ser Val Val Arg Oligonucleotide No. 5: cga aat gat aca cga GAA ttaagt gtt gtt cgt Arg Asn Asp Thr Arg Glu Leu Ser Val Val ArgOligonucleotide No. 6: ttg gct gat cga aat gaA TTT TTa aat tta agt gttgtt Leu Ala Asp Arg Asn Glu Phe Leu Asn Leu Ser Val Val OligonucleotideNo. 7: ttg gct gat cga aat gaA TTT TTa tta tta agt gtt gtt Leu Ala AspArg Asn Glu Phe Leu Leu Leu Ser Val Val Oligonucleotide No. 8: ttg gctgat cga aat gaA TTa TTa aat tta agt gtt gtt Leu Ala Asp Arg Asn Glu LeuLeu Asn Leu Ser Val Val Oligonucleotide No. 9: ttg gct gat cga aat gaATTa TTa tta tta agt gtt gtt Leu Ala Asp Arg Asn Glu Leu Leu Leu Leu SerVal Val Oligonucleotide No. 10: ttg gct gat cga aat gaA GAa GAa gaa ttaagt gtt gtt Leu Ala Asp Arg Asn Glu Glu Glu Glu Leu Ser Val ValOligonucleotide No. 11: ttg gct gat cga aat gaA GAa TTa tta tta agt gttgtt Leu Ala Asp Arg Asn Glu Glu Leu Leu Leu Ser Val Val OligonucleotideNo. 12: caa aat tgg ttg gct gaA TTa aat gaa tta tta aat Gln Asn Trp LeuAla Glu Leu Asn Glu Leu Leu Asn Oligonucieotide No. 13: caa aat tgg ttggct gaA TTa aat gaa ttt tta aat Gln Asn Trp Leu Ala Glu Leu Asn Glu PheLeu Asn Oligonucleotide No. 14: caa aat tgg ttg gct gaA TTa TTA gaa ttttta tta tta Gln Asn Trp Leu Ala Glu Leu Leu Glu Phe Leu Leu LeuOligonucleotide No. 15: caa aat tgg ttg gct gaA TTa TTA gaa tta tta ttatta Gln Asn Trp Leu Ala Glu Leu Leu Glu Leu Leu Leu Leu OligonucleotideNo. 16: caa aat tgg ttg gct gaA TTa TTA gaa gaa tta tta tta Gln Asn TrpLeu Ala Glu Leu Leu Glu Glu Leu Leu Leu Oligonucleotide No. 17: caa aattgg ttg gct gaA cga TTA gaa ttt tta tta tta Gln Asn Trp Leu Ala Glu ArgLeu Glu Phe Leu Leu Leu Oligonucleotide No. 18: caa aat tgg ttg gct gaAcga TTA gaa tta tta tta tta Gln Asn Trp Leu Ala Glu Arg Leu Glu Leu LeuLeu Leu Oligonucleotide No. 19: caa aat tgg ttg gct gaA TTa gaA gaa ttatta tta tta Gln Asn Trp Leu Ala Glu Leu Glu Glu Leu Leu Leu LeuOligonucleotide No. 20: caa aat tgg ttg gct gaA TTa TTA gaa gaa gaa gaatta

[0073] The successive site-directed mutagenesis procedure is similar tothe procedure described in Example 1. The difference lies in thecombination of oligonucleotides. For each of the examples of mutantsdescribed in Table 1, the successive combinations of oligonucleotidesare described below.

[0074] Mutant No. 1: The creation of mutant No. 1 requires twosuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 6 in the firstmutagenesis and oligonucleotide No. 13 in the second. OligonucleotideNo. 13 is defined to recognize the modifications introduced during thefirst mutagenesis with oligonucleotide No. 6.

[0075] Mutant No. 2: The creation of mutant No. 2 requires twosuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 8 in the firstmutagenesis and oligonucleotide No. 12 in the second. OligonucleotideNo. 12 is defined to recognize the modifications introduced during thefirst mutagenesis with oligonucleotide No. 8.

[0076] Mutant No. 3: The creation of mutant No. 3 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 7 in the second and oligonucleotide No.14 in the third. Oligonucleotide No. 7 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 14 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 7.

[0077] Mutant No. 4: The creation of mutant No. 4 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 9 in the second and oligonucleotide No.15 in the third. Oligonucleotide No. 9 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 15 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 9.

[0078] Mutant No. 5: The creation of mutant No. 5 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 11 in the second and oligonucleotideNo. 16 in the third. Oligonucleotide No. 11 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 16 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 11.

[0079] Mutant No. 6: The creation of mutant No. 6 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 7 in the second and oligonucleotide No.17 in the third. Oligonucleotide No. 7 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 17 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 7.

[0080] Mutant No. 7: The creation of mutant No. 7 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 9 in the second and oligonucleotide No.18 in the third. Oligonucleotide No. 9 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 18 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 9.

[0081] Mutant No. 8: The creation of mutant No. 8 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 4 in the firstmutagenesis, oligonucleotide No. 9 in the second and oligonucleotide No.19 in the third. Oligonucleotide No. 9 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 4 and oligonucleotide No. 19 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 4 and No. 9.

[0082] Mutant No. 9: The creation of mutant No. 9 requires threesuccessive series of site-directed mutagenesis according to the protocoldescribed in Example 1, using oligonucleotide No. 5 in the firstmutagenesis, oligonucleotide No. 10 in the second and oligonucleotideNo. 20 in the third. Oligonucleotide No. 10 is defined to recognize themodifications introduced during the first mutagenesis witholigonucleotide No. 5 and oligonucleotide No. 20 is defined to recognizethe modifications introduced during the first two mutageneses witholigonucleotides No. 5 and No. 10.

[0083] According to this protocol, the oligonucleotides are divided upinto three categories, 1st series oligonucleotides, 2nd seriesoligonucleotides and 3rd series oligonucleotides. This division is asfollows: 1st series oligonucleotides: oligonucleotides No. 4, 5, 6 and 82nd series oligonucleotides: oligonucleotides No. 7, 9, 10, 11, 12 and13 3rd series oligonucleotides: oligonucleotides No. 14, 15, 16, 17, 18,19 and 20.

[0084] The complete protocol for producing these mutants is identical tothat described in Example 1. This protocol is common to each of theseries of mutageneses, only the mutagenesis oligonucleotide and theoligonucleotide for inhibition/restoration of the resistance to theantibiotic change. The passing onto the following mutation takes placeafter screening of the clone of interest which has integrated thepreceding mutation. If this step is the final step of the first seriesor of the second series of mutagenesis, the material derived from thisseries of experiments is re-used as initial material for, respectively,the 2nd or 3rd series of mutagenesis using, respectively, the 2nd or 3rdseries oligonucleotides. A second cycle of mutagenesis can then becarried out using the plasmid DNA obtained as DNA matrix and also theoligonucleotide for repair of the tetracycline resistance gene and theoligonucleotide for destruction of the ampicillin resistance gene and a2nd series mutagenesis oligonucleotide. The recombinants are thenselected using medium supplemented with tetracycline at the finalconcentration of 12.5 μg/ml. A third cycle of mutagenesis can be carriedout using the plasmid DNA obtained at the end of the second cycle ofmutagenesis as DNA matrix and also the oligonucleotide for repair of theampicillin resistance gene and the oligonucleotide for destruction ofthe tetracycline resistance gene and a 3rd series mutagenesisoligonucleotide. The recombinants are then selected using mediumsupplemented with ampicillin at the final concentration of 100 μg/ml.After all the series of mutagenesis required to produce a mutant havebeen carried out, the steps for controlling the mutations are carriedout as described in Example 1.

Example 3 Creation of Pepsin Cleavage Sites in the α4-α5, α5-α6 andα6-α7 Inter-helix Loops of the Cry9Ca1 Toxin

[0085] The positions of the native sequences of the α4-α5, (α5-α6 andα6-α7 inter-helix loops of the Cry9Ca1 toxin are given in Table 2 below.The nucleotide sequences and the corresponding positions in the cry9Ca1gene are given in Table 3. TABLE 2 Position and sequences of the α4-α5,α5-α6 and α6-α7 inter-helix loops of the Cry9Ca1 toxin Loop SequencePosition Loop α4-α5 FAVNGQQVPLL Phenylalanine 187 to leucine 197 Loopα5-α6 LFGEGWGF Leucine 216 to phenylalanine 223 Loop α6-α7 LRGTN Leucine257 to asparagine 261

[0086] TABLE 3 Position and sequences of cry9Ca1 gene encoding theα4-α5, α5-α6 and α6-α7 inter-helix loops Loop Sequence Position Loopα4-α5 TTT GCA GTA AAT GGA CAG CAG GTT CCA TTA CTG 559-591 Loop α5-α6 CTTTTT GGA GAA GGA TGG GGA TTC 646-669 Loop α6-α7 TTA AGA GGA ACA AAT769-783

[0087] The superposition of the nucleotide and amino acid sequences areas follows: Loop TTT GCA GTA AAT GGA CAG CAG GTT CCA TTA CTG α4-α5: PheAla Val Asn Gly Gln Gln Val Pro Leu Leu Loop CTT TTT GGA GAA GGA TGG GGATTC α5-α6 Leu Phe Gly Glu Gly Trp Gly Phe Loop TTA AGA GGA ACA AAT α6-α7Leu Arg Gly Thr Asn

[0088] Pepsin-specific sites are introduced into the α4-α5, α5-α6 orα6-α7 inter-helix loops of the Cry9Ca1 toxin by substituting at leastone amino acid of these inter-helix loops with an amino acid recognizedby pepsin, namely leucine, phenylalanine and glutamic acid. Codonsencoding these three amino acids will therefore be created in place ofthe codons naturally present in the region extending from bases 559 to591 (α4-α5 inter-helix loop), 646 to 669 (α5-α6 inter-helix loop), and769 to 783 (α6-α7 inter-helix loop). The codon possibilities for thesethree amino acids are described in Example 1.

[0089] As in Example 1, the chosen organism for producing the modifiedCry protein is the B. thuringiensis bacterium, and the choice of thereplacement codons is therefore identical to that of Example 1. Inaddition, if another organism for production is chosen, those skilled inthe art will be able to adjust the preferential codons as a function ofthe chosen organism for production.

[0090] Various alternative sequences for the α4-α5, α5-α6 and α6-α7inter-helix loops are possible, each one having a variable number ofleucine, phenylalanine or glutamic acid residues. Several of thesevarious possibilities are given in Tables 4, 5 and 6. The possibilitiesfor modification of the α4-α5, α5-α6 and α6-α7 inter-helix loops are notlimited to those given in Tables 4, 5 and 6 below. The aim of the listgiven in Tables 4, 5 and 6 is to illustrate some of the possibilitiesfor modification without limiting the scope of the invention to theseillustrations. Those skilled in the art, being aware of the codonsspecific for each amino acid according to the organisms, will be able toadapt the teaching described in this example to all the possibilitiesfor modifying the α4-α5, α5-α6 and α6-α7 inter-helix loops, inparticular to those which are not described in Tables 4, 5 and 6. TABLE4 Examples of possible modifications of the α4-α5 inter-helix loop ofthe Cry9Ca1 toxin Protein Amino acid sequence Nucleotide sequence CryCa1FAVNGQVPLL ttt gca tga aat gga cag cag gtt cca tta ctg Phe Ala Val AsnGly Gln Gln Val Pro Leu leu Mutant No. 10 FLLNLFFLPLL ttt TTa Tta aatTTa TTT TTT TtA cca tta ctg Phe Leu leu Asn Leu Phe Phe Leu Pro Leu leuMutant No. 11 FLLNLEELPLL ttt TTa Tta aat TTa GaA GaA TtA cca tta ctgPhe Leu leu Asn Leu Glu Glu Leu Pro Leu Leu Mutant No. 12 FEENLEELPLLttt GAa GAa aat TTA GaA GaA TtA cca tta ctg Phe Glu Glu Asn Leu Glu GluLeu Pro Leu leu Mutant No. 13 FEENFLLFPLL ttt GAa GAa aat TTT TTA TTATtt cca tta ctg Phe Glu Glu Asn Phe leu Leu Phe Pro Leu leu Mutant No.14 FEENFEEFPLL ttt GAa GAa aat TTT GaA GaA Ttt cca tta ctg Phe Glu GluAsn Phe Glu Glu Phe Pro Leu leu Mutant No. 15 FLLNFEEFPLL ttt TTa TTaaat TTT GaA GaA Ttt cca tta ctg Phe Leu leu Asn Phe Glu Glu Phe Pro Leuleu Mutant No. 16 FLLNEFFEPLL ttt TTa TTa aat GAa TTT TTT gAA cca ttactg Phe Leu leu Asn Glu Phe Phe Glu Pro Leu leu

[0091] TABLE 5 Examples of possible modifications of the α5-α6inter-helix loop of the Cry9Cal toxin Amino acid Protein sequenceNucleotide sequence Cry9Ca1 LFGEGWGF ctt ttt gga gaa gga tgg gga ttc LeuPhe Gly Glu Gly Trp Gly Phe Mutant LFLELFLF ctt ttt TTa gaa TTa tTT TTattc No. 17 Leu Phe Leu Gly Leu Phe Leu Phe Mutant LFLLLFLF ctt ttt TTaTTa TTa tTT TTa ttc No. 18 Leu Phe Leu Leu Leu Phe Leu Phe MutantLFLEEFEL ctt ttt TTa gaa gAa tTT gAa TTA No. 19 Leu Phe Leu Glu Glu PheGlu Leu Mutant LFEEEFEL ctt ttt gAa gaa gAa tTT gAa TTA No. 20 Leu PheGlu Glu Glu Phe Glu Leu Mutant LFEEEFEE ctt ttt gAa gaa TTa tTT gAa GAANo. 21 Leu Phe Glu Glu Glu Phe Glu Glu

[0092] TABLE 6 Examples of possible modifications of the α6-α7inter-helix loop of the Cry9Ca1 toxin Amino acid Protein sequenceNucleotide sequence Cry9Ca1 LRGTN tta aga gga aca aat Leu Arg Gly thrAsn Mutant No. 22 LLELN tta TTa gAa TTa aat Leu Leu Glu Leu Asn MutantNo. 23 LLFLN tta TTa TTT TTa aat Leu Leu Phe Leu Asn Mutant No. 24 LELLNtta GAa TTa TTa aat Leu Glu Leu Leu Asn Mutant No. 25 LLFFN tta TTa TTTTTT aat Leu Leu Phe Phe Asn Mutant No. 26 LEELN tta GAa GAa TTa aat LeuGlu Glu Leu Asn Mutant No. 27 LEFLN tta GAa TTT TTa aat Leu Glu Phe LeuAsn Mutant No. 28 LEFEN tta GAa TTT GAa aat Leu Glu Phe Glu Asn MutantNo. 29 LEEEN tta GAa gAa GAa aat Leu Glu Glu Glu Asn

3-1-Creation of Pepsin Cleavage Sites in the α4-α5 Inter-helix Loop

[0093] The substitution of several amino acids within the α4-α5inter-helix loop requires, for each of the mutants, the successive useof several mutagenesis oligonucleotides. The mutagenesisoligonucleotides required to create the examples of mutants given inTable 4 are presented below (numbered from 21 to 34). OligonucleotideNo. 21: gct att cca ttg ttt TTa Tta aat gga cag cag gtt Ala Ile Pro LeuPhe Leu Ile Asn Gly Gln Gln Val Oligonucleotide No. 22: gct att cca ttgttt GAa GAa aat gga cag cag gtt Ala Ile Pro Leu Phe Glu Glu Asn Gly GlnGln Val Oligonucleotide No. 23: tta tta aat gga cag cag TtA cca tta ctgtca gta Leu leu Ann Gly Gln Gln Leu Pro Leu Leu Ser Val OligonucleotideNo. 24: tta tta aat gga cag cag Ttt cca tta ctg tca gta Leu leu Asn GlyGln Gln Phe Pro Leu Leu Ser Val Oiigonucleotide No. 25: tta tta aat ggacag cag gAA cca tta ctg tca gta Leu leu Asn Gly Gln Gln Glu Pro Leu LeuSer Val Oligonucleotide No. 26: gaa gaa aat gga cag cag TtA caa tta ctgtca gta Glu Glu Asn Gly Gln Gln Leu Pro Lou Leu Ser Val OligonucleotideNo. 27: gaa gaa aat gga cag cag Ttt cca tta ctg tca gta Glu Glu Asn GlyGln Gln Phe Pro Leu Leu Ser Val Oligonucleotide No. 28: cca ttg ttt ttatta aat TTa TTT TTT tta cca tta ctg tca gta Pro Lou Phe Lou Leu Asn LeuPhe Phe Leu Pro Leu Leu Ser Val Oligonucleotide No. 29: cca ttg ttt ttatta aat TTa GaA GaA tta cca tta ctg tca gta Pro Leu Phe Leu Leu Asn LeuGlu Glu Leu Pro Leu Leu Ser Val Oligonucleotide No. 30: cca ttg ttt gaagaa aat TTa GaA GaA tta cca tta ctg tca gta Pro Leu Phe Glu Glu Asn LeuGlu Glu Leu Pro Leu Leu Ser Val Oligonucleotide No. 31: cca ttg ttt gaagaa aat TTT TTA TTA ttt cca tta ctg tca gta Pro Leu Phe Glu Glu Asn PheLeu Leu Phe Pro Leu Leu Ser Val Oligonucleotide No. 32: cca ttg ttt gaagaa aat TTT GaA GaA ttt cca tta ctg tca gta Pro Leu Phe Glu Glu Asn PheGlu Glu Phe Pro Leu Leu Ser Val Oligonucleotide No. 33: cca ttg ttt ttatta aat TTT GaA GaA ttt cca tta ctg tca gta Pro Leu Phe Leu Leu Asn PheGlu Glu Phe Pro Leu Leu Ser Val Oligonucleotide No. 34: cca ttg ttt ttatta sat GAa TTT TTT gaa cca tta ctg tca gta Pro Leu Phe Leu Leu Asn GluPhe Phe Glu Pro Leu Leu Ser Val

[0094] The successive site-directed mutagenesis procedure is similar tothe procedure described in Example 2. The only difference lies in thecombination of oligonucleotides. For each of the mutants described inTable 4, the successive combinations of oligonucleotides are describedbelow.

[0095] Mutant No. 10: The creation of mutant No. 10 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 21 in the first mutagenesis,oligonucleotide No. 23 in the second and oligonucleotide No 28 in thethird. Oligonucleotide No. 23 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 21 andoligonucleotide No. 28 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 21and 23.

[0096] Mutant No. 11: The creation of mutant No. 11 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 21 in the first mutagenesis,oligonucleotide No. 23 in the second and oligonucleotide No 29 in thethird. Oligonucleotide No. 23 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 21 andoligonucleotide No. 29 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 21and 23.

[0097] Mutant No. 12: The creation of mutant No. 12 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 22 in the first mutagenesis,oligonucleotide No. 26 in the second and oligonucleotide No 30 in thethird. Oligonucleotide No. 26 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 22 andoligonucleotide No. 30 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 22and 26.

[0098] Mutant No. 13: The creation of mutant No. 13 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 22 in the first mutagenesis,oligonucleotide No. 27 in the second and oligonucleotide No 31 in thethird. Oligonucleotide No. 27 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 22 andoligonucleotide No. 31 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 22and 27.

[0099] Mutant No. 14: The creation of mutant No. 14 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 22 in the first mutagenesis,oligonucleotide No. 27 in the second and oligonucleotide No 32 in thethird. Oligonucleotide No. 27 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 22 andoligonucleotide No. 32 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 22and 27.

[0100] Mutant No. 15: The creation of mutant No. 15 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 21 in the first mutagenesis,oligonucleotide No. 24 in the second and oligonucleotide No 33 in thethird. Oligonucleotide No. 24 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 21 andoligonucleotide No. 33 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 21and 24.

[0101] Mutant No. 16: The creation of mutant No. 16 requires threesuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 21 in the first mutagenesis,oligonucleotide No. 25 in the second and oligonucleotide No 34 in thethird. Oligonucleotide No. 25 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 21 andoligonucleotide No. 34 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 21and 25.

[0102] According to this protocol, the oligonucleotides intended tocreate the mutants No. 10 to No. 16 described in Table 4 are divided upinto three categories, 1st series oligonucleotides, 2nd seriesoligonucleotides and 3rd series oligonucleotides. This division is asfollows: 1st series oligonucleotides: oligonucleotides No. 21 and 22 2ndseries oligonucleotides: oligonucleotides No. 23, 24, 25, 26 and 27 3rdseries oligonucleotides: oligonucleotides No. 28, 29, 30, 31, 32, 33 and34.

3-2-Creation of Pepsin Cleavage Sites in the α5-α6 Inter-helix Loop

[0103] The substitution of several amino acids within the α5-α6inter-helix loop requires, for each of the mutants, the successive useof several mutagenesis oligonucleotides. The mutagenesisoligonucleotides required to create the examples of mutants given inTable 5 are presented below (numbered from 35 to 44). OligonucleotideNo. 35: gat gca tct ctt ttt TTa gaa gga tgg gga ttc Asp Ala Ser Leu PheLeu Glu Gly Trp Gly Phe Ollgonucleotide No. 36: gat gca tct ctt ttt TTaTTa gga tgg gga ttc aca Asp Ala Ser Leu Phe Leu Leu Gly Trp Gly Phe ThrOligonucleotide No. 37: gat gca tct ctt ttt gAa gaa gga tgg gga ttc AspAla Ser Leu Phe Glu Glu Gly Trp Gly Phe Oligonucleotide No. 38: tta gaagga tgg gga TTa aca sag ggg gaa att Leu Glu Gly Trp Gly Leu Thr Gln GlyGlu Ile Oligonucleotide No. 39: gga gaa gga tgg gga GAA aca sag ggg gaaatt Gly Glu Gly Trp Gay Glu Thr Gln Gay Glu Ile Oligonucleotide No. 40:gca tct ctt ttt tta gaa TTa tTT TTa ttc aca cag gqg gaa att Ala Ser LeuPhe Leu Glu Leu Phe Leu Phe Thr Gln Gly Glu Ile Oligonucleotide No. 41:gca tot ctt ttt tta tta TTa tTT TTa ttc aca cag ggg gaa att Ala Ser LeuPhe Leu Leu Leu Phe Leu Phe Thr Gln Gly Glu Ile Oligonucleotide No. 42:gca tct ctt ttt tta gaa TTa tTT TTa ttc aca cag ggg gaa att Ala Ser LeuPhe Leu Glu Glu Phe Gln Leu Thr Gln Gly Glu Ile Qligonucleotide No. 43:gca tct ctt ttt gaa gaa TTa tTT TTa ttc aca cag ggg gaa att Ala Ser LeuPhe Glu Glu Glu Phe Glu Leu Thr Gln Gly Glu Ile Oligonucleotide No. 44:gca tct ctt ttt gaa gaa TTA tTT TTa gaa aca cag ggg gaa att Ala Ser LeuPhe Glu Glu Glu Phe Glu Glu Thr Gln Gly Glu Ile

[0104] The successive site-directed mutagenesis procedure is similar tothe procedure described in Example 2. The only difference lies in thecombination of oligonucleotides. For each of the mutants described inTable 5, the successive combination of oligonucleotides are describedbelow.

[0105] Mutant No. 17: The creation of mutant No. 17 requires twosuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 35 in the first mutagenesisand oligonucleotide No. 40 in the second. Oligonucleotide No. 40 isdefined to recognize the modifications introduced during the firstmutagenesis with oligonucleotide No. 35.

[0106] Mutant No. 18: The creation of mutant No. 18 requires twosuccessive series of site-directed mutageneses according to the protocoldescribed below, using oligonucleotide No. 36 in the first mutagenesisand oligonucleotide No. 41 in the second. Oligonucleotide No. 41 isdefined to recognize the modifications introduced during the firstmutagenesis with oligonucleotide No. 36.

[0107] Mutant No. 19: The creation of mutant No. 19 requires threesuccessive series of site-directed mutageneses according to the protocolbelow, using oligonucleotide No. 35 in the first mutagenesis,oligonucleotide No. 38 in the second and oligonucleotide No. 42 in thethird. Oligonucleotide No. 38 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 35 andoligonucleotide No. 42 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 35and 38.

[0108] Mutant No. 20: The creation of mutant No. 20 requires threesuccessive series of site-directed mutageneses according to the protocolbelow, using oligonucleotide No. 37 in the first mutagenesis,oligonucleotide No. 38 in the second and oligonucleotide No 43 in thethird. Oligonucleotide No. 38 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 37 andoligonucleotide No. 43 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 37and 38.

[0109] Mutant No. 21: The creation of mutant No. 21 requires threesuccessive series of site-directed mutageneses according to the protocolbelow, using oligonucleotide No. 37 in the first mutagenesis,oligonucleotide No. 39 in the second and oligonucleotide No 44 in thethird. Oligonucleotide No. 39 is defined to recognize the modificationsintroduced during the first mutagenesis with oligonucleotide No. 37 andoligonucleotide No. 44 is defined to recognize the modificationsintroduced during the first two mutageneses with oligonucleotides No. 37and 39.

[0110] According to this protocol, the oligonucleotides intended tocreate mutants No. 17 to No. 21 described in Table 5 are divided up intothree categories, 1st series oligonucleotides, 2nd seriesoligonucleotides and 3rd series oligonucleotides. This division is asfollows: 1st series oligonucleotides: oligonucleotides No. 35, 36 and 372nd series oligonucleotides: oligonucleotides No. 38, 39, 40 and 41 3rdseries oligonucleotides: oligonucleotides No. 42, 43 and 44.

3-3-Creation of Pepsin Cleavage Sites in the α6-α7 Inter-helix Loop

[0111] The substitution of several amino acids within the α6-α7inter-helix loop requires, for each of the mutants, only onemutagenesis. The mutagenesis oligonucleotides required to create theexamples of mutants given in Table 6 are presented below (numbered from45 to 52). Oligonucleotide No. 45: ggt tta gat cgt tta TTa gAa TTa aatact gaa agt tgg Gly Leu Asp Arg Leu Leu Glu Leu Asn Thr Glu Ser TrpOligonucleotide No. 46: ggt tta gat cgt tta TTa TTT TTa aat act gaa agttgg Gly Leu Asp Arg Leu Leu Phe Leu Asn Thr Glu Ser Trp OligonucleotideNo. 47: ggt tta gat cgt tta GAa TTa TTa aat act gaa agt tgg Gly Leu AspArg Leu Glu Leu Leu Asn Thr Glu Ser Trp Oligonucleotide No. 48: ggt ttagat cgt tta TTa TTT TTT aat act gaa agt tgg Gly Leu Asp Arg Leu Leu PhePhe Asn Thr Glu Ser Trp Oligonucleotide No. 49: ggt tta gat cgt tta GAaGAa TTa aat act gaa agt tgg Gly Leu Asp Arg Leu Glu Glu Leu Asn Thr GluSer Trp Oligonucleotide No. 50: ggt tta gat cgt tta GAa TTT TTa aat actgaa agt tgg Gly Leu Asp Arg Leu Glu Phe Leu Asn Thr Glu Ser TrpOligonucleotide No. 51: ggt tta gat cgt tta GAa TTT GAa aat act gaa agttgg Gly Leu Asp Arg Leu Glu Phe Glu Asn Thr Glu Ser Trp OligonucleotideNo. 52: ggt tta gat cgt tta GAa gAa GAa aat act gaa agt tgg Gly Leu AspArg Leu Glu Glu Gln Asn Thr Glu Ser Trp Oligonucleotide No. 45 is usedto create mutant No. 22. Oligonucleotide No. 46 is used to create mutantNo. 23. Oligonucleotide No. 47 is used to create mutant No. 24.Oligonucleotide No. 48 is used to create mutant No. 25. OligonucleotideNo. 49 is used to create mutant No. 26. Oligonucleotide No. 50 is usedto create mutant No. 27. Oligonucleotide No. 51 is used to create mutantNo. 28. Oligonucleotide No. 52 is used to create mutant No. 29.

[0112] The complete protocol for producing these mutants is identical tothat described in Example 2. This protocol is common to each of theseries of mutageneses, only the mutagenesis oligonucleotide and theoligonucleotide for inhibition/restoration of the resistance to theantibiotic change.

Example 4: Creation of Pepsin Cleavage Sites in the α3-α4, α4-α5, α5-α6and α6-α7 Inter-helix Loops of Various Cry Toxins

[0113] Several groups of Cry proteins exhibit structural similarities.They are in particular the proteins belonging to the Cry1, Cry3, Cry4,Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 or Cry20 families. Thesesimilarities are demonstrated in the literature (Schnepf et al., 1998).Other Cry proteins not cited in the literature can also exhibitstructural and sequence similarities with these families. The aim ofExample 4 is to demonstrate the applicability of the teaching of thepresent invention, as exemplified on the Cry9Ca1 protein in Examples 2and 3, to all these structurally similar families.

[0114] The modifications in the inter-helix loops described in Examples2 and 3 can be carried out in an equivalent manner for all the Cryproteins in which it is possible to identify inter-helix loops similarto those present in domain I of the Cry9Ca1 toxin. If the location andthe sequence of these inter-helix loops are defined for these variousCry toxins, it is very easy for those skilled in the art to formmodifications similar to those given in Examples 2 and 3 using thetechnical details provided in these same Examples 2 and 3. In thepresent example, the elements for creating specific sites fordegradation by pepsin in the Cry toxins other than the Cry9Ca1 toxin,and in particular the Cry1, Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16,Cry17, Cry19 and Cry20 proteins, are given. The modification of theseinter-helix loops to create sites for degradation by pepsin in the Cry1,Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 or Cry20 toxinsrequires the following steps to be followed:

[0115] 1) Establish, according to the sequences and the locations of theinter-helix loops given in Tables 6-13 below, lists of possible mutantshaving one or more leucine, phenylalanine or glutamic acid residues asgiven in Tables 1, 4, 5 and 6 and in Examples 2 and 3.

[0116] 2) Establish the sequences of the mutant genes taking intoaccount the codon preference of the host organism and, if this organismis B. thuringiensis, preferentially using the codons TTA, TTT and GAAfor leucine, phenylalanine and glutamic acid, respectively.

[0117] 3) Synthesizing mutagenesis oligonucleotides for modifying thesequence of the genes encoding the toxins selected based on the modelfor those given in Examples 2 and 3.

[0118] 4) Use single or multiple mutagenesis strategies as described inExamples 2 and 3 and according to the experimental protocols describedin detail in Examples 2 and 3.

[0119] The location of the α3-α4, α4-α5, α5-α6 and α6-α7 inter-helixloops of domain I and their sequences are given, for the Cry1, Cry3,Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20 toxins, inTables 7, 8, 9, 10, 11, 12 and 13 below. These sequences are given foreach of the holotype proteins as defined by the Bacillus thuringiensisclassification committee (Crickmore et al., 2001). However, since theintra-holotype sequence homologies, i.e. the sequence homologies betweenthe various subtypes of the same holotype, are very high, those skilledin the art will be able to adapt the teaching of the present Example 4to all the Cry protein subtypes. TABLE 7 Location and sequence of theα3-α4 inter-helix loop in the Cry1 proteins Amino acid Position inNucleotide Protein sequence protein sequence Position in gene CrylAaDPTN 120 to 123 gatcctactaat 358 to 369 CrylAb DPTN 120 to 123gatcctactaat 358 to 369 CrylAc DPTN 120 to 123 gatcctactaat 358 to 369CrylAd DPTN 120 to 123 gatcctactaat 358 to 369 CrylAe DPTN 120 to 123gatcctactaat 358 to 369 CrylAf DPTN 120 to 123 gatcctactaat 358 to 369CrylAg DPTN 120 to 123 gatcctactaat 358 to 369 CrylBa NRDD 139 to 142aaccgtgatgat 415 to 426 CrylBb NRND 144 to 147 aaccgaaatgat 430 to 441CrylBc NRND 144 to 147 aaccgaaatgat 430 to 441 CrylBd NRND 144 to 147aaccgaaatgat 430 to 441 CrylCa DPNN 119 to 122 gatcctaataat 355 to 366CrylCb DPDN 119 to 122 gatcctgataat 355 to 366 CrylDa DPTN 119 to 122gatcctactaat 355 to 366 CrylDb DPSN 119 to 122 gatccgtctaat 355 to 366CrylEa DPTN 118 to 121 gatcctactaat 352 to 363 CrylEb DPTN 117 to 120gatcctactaat 349 to 360 CrylFa NPNN 118 to 121 aatcctaataat 352 to 363CrylFb NPNN 118 to 121 aatcctaataat 352 to 363 CrylGa DPNN 118 to 121gatcctaataat 352 to 363 CrylGb DPDN 118 to 121 gatcctgataac 352 to 363CrylHa SPNN 122 to 125 tctcctaataat 364 to 375 CrylHb SPNN 121 to 124tctcctaataat 361 to 372 CrylIa NRNN 148 to 151 aatcgtaataac 442 to 453CrylIb NRNN 148 to 151 aatcgtaataac 442 to 453 CrylIc NRNN 148 to 151aatcgtaataac 442 to 453 CrylId NRNN 148 to 151 aatcgcaataac 442 to 453CrylIe NRNN 148 to 151 aatcgcaacaac 442 to 453 CrylJa DPDN 119 to 122gatcctgataac 355 to 366 CrylJb TPDN 119 to 122 actccagataac 355 to 366CrylKa NRND 145 to 148 aaccgaaatgat 433 to 444

[0120] TABLE 8 Location and sequence of the α4-α5 inter-helix loop inthe Cry1 proteins Amino acid Position in Protein sequence proteinNucleotide sequence Position in gene Cry1Aa LAVQNYQVPLL 148 to 158ttggcagttcaaaattatcaagttcctctttta 442 to 474 FLAVQNYQVPLL 148 to 158ttgcagttcaaaattatcaagttcctctttta 442 to 474 Cry1Ab FAVQNYQVPLL 148 to158 ttgcagttcaaaattatcaagttcctctttta 442 to 474 Cry1Ac FAVQNYQVPLL 148to 158 tttgcagttcaaaattatcaagttcctctttta 442 to 474 LAVQNYQVPLL 148 to158 ttggcagtcaaaattatcaagttcctctttta 442 to 474 Cry1Ad FTVQNYQVPLL 148to 158 cctacagttcaaaattatcaagtacctcttcta 442 to 474 Cry1Ae FTVQNYQVPLL148 to 158 tttacagttcaaaattatcaagtacctcttcta 442 to 474 Cry1AfFAVQNYQVPLL 148 to 158 tttgcagttcaaaattatcaagttcctctttta 442 to 474Cry1Ag LAVQNYQVPLL 148 to 158 ttggcagttcaaaattatcaagttcctctttta 442 to474 Cry1Ba FAIRNQEVPLL 167 to 177 ttcgcaattagaaaccaagaagttccattattg 499to 531 Cry1Bb FRIRNEEVPLL 172 to 182 ttcagaatacgaaatgaagaagttccattatta514 to 546 Cry1Bc FRIRNEEVPLL 172 to 182ttcagaatacgaaatgaagaagttccattatta 514 to 546 Cry1Bd FRIRNEEVPLL 172 to182 ttcagaatacgaaatgaagaagttccattatta 514 to 546 Cry1Ca FRISGFEVPLL 147to 157 tttcgaatttctggatttgaagtacccctttta 439 to 471 Cry1Cb FRIAGFEVPLL147 to 157 tttcgaattgctggatttgaagtacccctttta 439 to 471 Cry1DaFRVQNYEVALL 147 to 157 tttagagttcaaaattatgaagttgctctttta 439 to 471Cry1Db LRVRNYEVALL 147 to 157 ttaagagttcgtaattatgaagttgctctttta 439 to471 Cry1Ea LFSVQNYQVPFL 145 to 156 aattttacacttacaagttttgaaatccctctttta433 to 468 Cry1Fb NFTLTSFEIPLL 145 to 156aattttacacttacaagttttgaaatccctctttta 433 to 468 Cry1Ga RLAIRNLEVVNL 145to 156 actttggcaattcggaatcttgaggtagtgaattta 433 to 468 Cry1GbLMAIPGPELATL 145 to 156 cttatggcaattccaggtttgaattagctactttta 433 to 468Cry1Gb LMAIPGPELATL 145 to 156 cttatggcaattccaggttttgaattagctacttta 433to 468 Cry1Ha LREQGFEIPLL 150 to 160 ctgagagaacaaggctttgaaattcctctttta448 to 480 Cry1Hb LREQGFEIPLL 149 to 159ctgagagaacagggctttgaaattcctctttta 445 to 477 Cry1Ia FAVSGEEVPLL 176 to186 tttgcagtgtctggagaggaggtaccattatta 526 to 558 Cry1Ib FAVSGEEVPLL 176to 186 tttgcagtatctggtgaggaagtaccattatta 526 to 558 Cry1Ic FAVSGEEVPLL176 to 186 tttgcagtatctggtgaggaagtaccattatta 526 to 558 Cry1IdFAVSGEEVPLL 176 to 186 tttgcagtttctggagaagaggtgccgctatta 526 to 558Cry1Ie FAVSGEEVPLL 176 to 186 tttgcagtatcaggtgaggaagtaccattattg 526 to558 Cry1Ja FRIIGFEVPLL 147 to 157 tttcggataattggatttgaagtgccactttta 439to 471 Cry1Jb FRIPGFEVPLL 147 to 157 tttcggattcccggatttgaagtgccacttcta439 to 471 Cry1Ka FSIRNEEVPLL 173 to 183ttcagcatacgaacgaagaggttccattattta 517 to 549

[0121] TABLE 9 Location and sequence of the α5-α6 inter-helix loop inthe Cry1 proteins Amino acid Position in Protein sequence proteinNucleotide sequence Position in gene Cry1Aa FGQRWGFD 178 to 185tttggacaaaaggtggggatttgat 532 to 555 Cry1Ab FGQRWGFD 178 to 185tttggacaaaggtggggatttgat 532 to 555 Cry1Ac FOQRWGPD 178 to 185tttggacaaaggtggggatttgat 532 to 555 Cry1Ad FGQRWGFD 178 to 185ttggacaacgttggggatttgat 532 to 555 Cry1Ae FGQRWGLD 178 to 185tttggacaacgttggggacttgat 532 to 555 Cry1Af CGQRSGFD 175 to 182tgtggacaaaggtcgggatttgat 523 to 546 Czy1Ag FGQRWGFD 178 to 185tttggacaaaggtggggatttgat 532 to 555 Cry1Ba FGSEFGLT 197 to 204ttggtagtgaatttgggcttaca 589 to 612 Cry1Bb FGSEWGMA 202 to 209tttggtagtgaatgggggatggca 604 to 627 Cey1Bc FGSEWGMA 202 to 209tttggtagtgaatgggggatggca 604 to 627 Cry1Bd FGSEWGMA 202 to 209tttggtagtgaatgggggatggca 604 to 627 Cry1Ca FGERWGLT 177 to 184ttggagaaagatggggattgaca 529 to 552 FGERWGVT 177 to 184ttggagaaagatggggagtgaca 529 to 552 Cry1Cb FGARWGLT 177 to 184tttggagcaagatggggattgaca 529 to 552 Cry1Da FGERWGYD 177 to 184ttcggagaaagatggggatatgat 529 to 552 Cry1Db YGQRWGFD 177 to 184tacggtcagagatggggctttgac 529 to 552 Cry1Ea FGQAWGFD 176 to 183tttgggcaggcttggggatttgat 526 to 549 Cry1Eb FGQRWGFD 175 to 182tttggacaacgttggggatttgat 523 to 546 Cry1Fa FGQGWGLD 176 to 183tttgggcagggttggggactggat 526 to 549 Cry1Fb FGQGWGLD 176 to 183tttgggcagggttggggctggat 526 to 549 Cry1Ga FGERWGLT 176 to 183tttggagaaagatggggattaaca 526 to 549 Cry1Gb FGERWGLT 176 to 183tttggggagagatggggattgaca 526 to 549 Cry1Ha FGQRWGLD 180 to 187tttgggcaaagatggggacttgac 538 to 561 Cry1Hb FGQRWGLD 179 to 186tttggacagagatggggacttgat 535 to 558 Cry1Ia FGKEWGLS 206 to 213tttggaaaagagtggggattatca 616 to 639 Cry1Ib FGKEWGLS 206 to 213tttgaaagaatggggattatca 616 to 639 Cry1Ic FEKNGGLS 206 to 213ttgaaaagaatgggggattatca 616 to 639 Cry1Id FGKEWGLS 206 to 213tttggaaaagaatgggggattgtca 616 to 639 Cry1Ie FGKEWGLS 206 to 213tttggaaaagagtggggattatct 616 to 639 Cry1Ja FGERWGLT 177 to 184ttggagagagatggggattgacg 529 to 552 Cry1Jb FGERWGLT 177 to 184ttcggagagagatggggattgacg 529 to 552 Cry1Ka FGSEWGMS 203 to 210tttggtagtgaatgggggatgtca 607 to 630

[0122] TABLE 10 Location and sequence of α6-α7 inter-helix loop in theCry1 proteins Amino acid Position in Protein sequence protein Nucleotidesequence Position in gene Cry1Aa VWGPD 218 to 222 gtatggggaccggat 652 to666 Cry1Ab VWGPD 218 to 222 gtatggggaccggat 652 to 666 Cry1Ac VWGPD 218to 222 gtatggggaccggat 652 to 666 Cry1Ad VWGPD 218 to 222gtatggggaccggaa 652 to 666 Cry1Ae VWGPD 218 to 222 gtatggggaccggat 652to 666 Cry1Af VWGPD 215 to 219 gtatggggaccggat 643 to 657 Cry1Ag VWGPD218 to 222 gtatggggacecgac 652 to 666 Cry1Ba LRGTN 237 to 241ttgagagggacaaaa 709 to 723 Cry1Bb LRGTN 242 to 246 ttaagagggacaaat 724to 738 Cry1Bc LRGTN 242 to 246 ttaagagggacaaat 724 to 738 CrylBd LRGTN242 to 246 ttaagagggacaaat 724 to 738 Cry1Ca LPKST 217 to 221ttaccgaaatctacg 649 to 663 Cry1Cb LPKST 217 to 221 ttaccaaaatcracg 649to 663 Cry1Da LEGRF 217 to 221 ttaggaaggtcgtttt 649 to 663 Cry1Db LEGSR217 to 221 ttagagggatctcga 649 to 663 Cry1Ea LPRTGG 216 to 221ttaccacgaactggtggg 646 to 663 Cry1Eb LPRNEG 215 to 220ttaccacgtaatgaaggg 643 to 660 CrylFa LRGTNT 216 to 221ataagaggtaataatact 646 to 663 Cry1Fb LRGTNT 216 to 221ttaagaggtactaatact 646 to 663 Cry1Ga IGGIS 216 to 220 attggagggataagt646 to 660 Cry1Gb LNVIR 216 to 220 ttaaatgttataaga 646 to 660 Cry1HaFGGVS 220 to 224 tttggtggtgtgtca 658 to 672 Cry1Hb FGVVT 219 to 223tttggtgttgtaaca 655 to 669 Cry1Ia LRGTN 246 to 250 ttgaggggtacaaat 736to 750 Cry1Ib LRGTN 246 to 250 ttgaggggtacaaat 736 to 750 Cry1Ic LRATN246 to 250 ttgagggctacaaat 736 to 750 Cry1Id LRGTN 246 to 250ttgaggggaacaaat 736 to 750 Cry1Ie LRGTN 246 to 250 ttgagaggtacaaat 736to 750 Cry1Ja LGPRS 217 to 221 cagggtttagatct 649 to 663 Cry1Jb LGFTS217 to 221 ctagggtttacttct 649 to 663 Cry1Ka LRGTT 243 to 247ttaagagggacaact 727 to 741

[0123] TABLE 11 Location and sequence of the α3-α4 inter-helix loop inthe Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20proteins Amino acid Position in Protein sequence protein Nucleotidesequence Position in gene Cry3Aa NPVSSRN 153 to 159aatcctgtgagulcacgaaat 457 to 477 Cry3Ba APVNLRS 154 to 160gcgcctgtaaatttacgaagt 460 to 480 Cry3Eb TPLSLRS 154 to 160acacctttaagtttgcgaagt 440 to 480 Cry3Ca TPLTLRD 151 to 157actcctttgactttcgagat 451 to 471 Cry4Aa NNPNPQNTQD 160 to 169aaataatcaaacccacaaaatactcaggat 478 to 501 Cry4Ba EPNNQSYRTA 136 to 145gagcctaataaaccagtcctatagaacagca 406 to 435 Cry7Aa KQDDPEAILS 147 to 156aaacaagatgatccagaagctatactttct 439 to 468 Cry7Ab NPDDPATITR 147 to 156aatcctgatgaccagcaactataacacga 439 to 468 Cry8Aa NRNDARTRSV 158 to 167aatcgcaatgatgcaagaactaagtgtt 472 to 501 Czy8Ba NPNGSRALRD 159 to 168aatccaaatggttcaagagccttacgagat 415 to 504 Cry8Ca NPHSTRSAAL 159 to 168aacccacacagtacacgaagcgcagcactt 475 to 504 Cry9Aa NPNSASAEEL 146 to 155aatcctaattctgcttctgctgaagaactc 436 to 465 Cry9Ba RPNGVRANLV 134 to 143agaccaacggcgtaagagcaaacttagtt 400 to 429 Cry9Ca DRNDTRNLSV 159 to 168gatcgaaacgatacacgaaatttaagtgtt 475 to 504 Cry9Da RPNGARASLV 159 to 168agaccaaatggcgcaagggcatccttagtt 475 to 504 Cry9Ea RPNGARANLV 159 to 168agaccgaacggagcaagagctaacttagtt 475 to 504 Cry10Aa ARTHANAKAV 162 to 171gcacgtacacacgctaatgctaaagcagta 484 to 513 Cry16Aa NYNPTSIDDV 109 to 118aattataatccaacttctatagacgatgta 325 to 354 Cry17Aa NKDDPLAIAEL 127 to 131aataaagatgaccccttggctatagctgaatta 379 to 411 Cry19Aa DPKSTGNLSTL 159 to169 gatccaaaatacaggtaatttaagcacctta 475 to 507 Cry19Ba NKNNFASGEL 151 to160 aataaaaataatttcgcaagtggtgaactt 451 to 480 Cry20Aa ERNRTRENGQ 141 to150 gaacgtaatagaactcgtgaaaacggacaa 421 to 450

[0124] TABLE 12 Location and sequence of the α4-α5 inter-helix loop inthe Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20proteins Amino acid Position Position Protein sequence in proteinNucleotide sequence in gene Cry3Aa ISGYEVL 186 to 192atttctggatacgaggttcta 556 to 576 CRy3Ba VSKFEVL 157 to 193gtttccaaattcgaagttctg 559 to 579 CRY3Bb VSKFEVL 187 to 193gtttccaaattcgaagtgctg 559 to 579 Cry3Ca VSGYEVL 184 to 190gtctctggatacgaagttcta 550 to 570 Cry4Aa LVNSCPPNPSDCDYYNILVL 188 to 207cttgtaaactcttgtcctcctaatcctagtgattgcgattactataacat 562 to 621 actagtattaCry4Ba FSNLVGYELLLL 164 to 175 tttagcaacttagtaggttatgaattattgttatta 490to 525 Cry7Aa FKVTGYEIPLL 175 to 185 tttaaggttactggatatgaaataccattacta523 to 555 Cry7Ab FRVAGYEIPLL 175 to 185tttagggttgctggatatgaaataccattacta 523 to 555 Cry8Aa FAVSGHEVLLL 136 to196 tttgcagtatccggacacgaagtactattatta 556 to 588 Cry8Ba FRVTNFEVPFL 187to 197 tttcgagtgacaaauttgaagtaccatttcctt 559 to 591 Cry8Ca FSQTNYETPLL187 to 197 ttttacaaacgaattatgagactccactctta 559 to 591 Cry9AaLTNGGSLARQNAQILLL 175 to 191ttaacgaatggtggtctgttagctagacaaaatgcccaaatattattatt 523 to 571 a Cry9BaFGSGPGSQRFQAQLL 161 to 175 tttggtagtggccctggaagtcaaaggtttcaggcacaatgttg481 to 525 Cry9Ca FAVNGQQVPLL 187 to 197tttgcagtaaatggcacagcaggttccattactg 559 to 591 Cry9Da FGSGPGSQRJYATILL116 to 200 tttggctctggtcctggaagtcaaaattatgcaactatattactt 556 to 600Cry9Ea FGTOPOSQIWAVALL 186 to 200tttggtacgggtcctggtagtcaaagagatgcggtagcgttgttg 556 to 600 Cry10AaLKNNASYRIPTL 189 to 200 ttaaaaaataatgctagctatcgfaataccaacactc 565 to 600Cry16Aa FKVKNYEVTVL 136 to 146 tttaaggttaaaaattatgaagtaacagtgtta 406 to438 Cry17Aa FKRANYEVLLL 155 to 165 tttaaaagggcgaattatgaagtcttactatta 463to 495 Cry19Aa VNNQOSPOYELLLL 187 to 200gttaaataatcaggggagtccaggttatgagttacttttattg 559 to 600 Cry19BaFSLGGYETVLL 180 to 190 ttctcattaggaggttatgaaacagtattatta 538 to 570Cry20Aa LSRRGFETLLL 173 to 183 ctttctcgcagaggattcgaaactcttttatta 517 to549

[0125] TABLE 13 Location and sequence of the α5-α6 inter-helix loop inthe Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20proteins Amino acid Position in Protein sequence protein Nucleotidesequence Position in gene Cry3Aa GEEWGYE 215 to 222ggagaagantggggatacgaa 643 to 663 Cry3Ba GEEWGYS 216 to 222ggagaagaatggggatattct 646 to 666 Cry3Bb GEEWGYS 226 to 222ggagaagaatggggatattct 646 to 666 Cry3Ca GTDWGYS 213 to 219ggaacggattggggatattct 637 to 657 Cry4Aa FEAYLKNNRQFDYEL 227 to 241tttgaagcgtatttaaaaaacaattcgattattttagag 679 to 723 Cry4Ba LINAQEWSL 193to 201 ctcataaatgcacaagaatggtcttta 577 to 603 PHKCTRMVY 193 201cctcataaatgcacaagaatggtctat 577 to 603 Cry7Aa GDKWGF 206 to 211ggagataaatggggattc 616 to 633 GDKWEF 206 to 221 ggagataaatgggaattc 616to 633 Cry7Ab GDKWGF 206 to 211 ggagataaatgggguttc 616 to 633 Cry8AaGEEWGF 217 to 222 ggagaagagtggggattt 649 to 666 Cry8Ba GEEWGL 218 to 223ggagaagaatggggattg 652 to 669 Cry8Ca GKEWGY 218 to 223gggaaggaatggggatat 652 to 669 Cry9Aa RYGTNWGL 210 to 217agatatggcactaattgggggcta 628 to 651 Cry9Ba1 KYGARWGL 194 to 201aagtatggggcaagatggggatc 580 to 603 Cry9Ca LFGEGWGF 216 to 223ctttttggagaaggatggggattc 646 to 669 Cry9Da IYGARWGL 219 to 226atttatggagcagatgggggctg 655 to 678 Cry9Ea IYGARWOL 219 to 226atctaggggcaagatggggactt 655 to 678 Cry10Aa TYYNIWLQ 219 to 226acttatggggcaagatggggactt 655 to 673 Cry16Aa IYGDAWNLYRELGP 265 to 178atttatggagatgcatggaatttatatagagaattaggattt 493 to 534 Cry17Aa LLNKVIDNF184 to 192 cttttaaataagttatagataatttt 550 to 576 Cry19Aa IYGDKWWSA 219to 227 atttatggagataaatggtggagcgca 655 to 681 Cry19Ba IYGKELG 209 to 215atttacggaaaagaattagga 625 to 645 Cry20Aa LYRNQWL 202 to 208ctttatagaaatcaatggtta 604 to 624

[0126] TABLE 14 Location and sequence of the α6-α7 inter-helix loop inthe Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20proteins Amino acid Position in Protein sequence protein Nucleotidesequence Position in gene Cry3Aa RGSS 255 to 258 agaggttcatct 763 to 774Cry3Ba RGST 256 to 259 agaggttcaact 766 to 777 Cry3Bb RGST 256 to 259agaggttcaat 766 to 777 Cry3Ca RGST 253 to 256 agaggttcgact 757 to 768Cry4Aa LIKTTPD 274 to 280 ttaattaaaacgacgcctgat 820 to 840 Cry4Ba LRNKS235 to 239 cttagaaataaatct 703 to 717 Cry7Aa LNGST 245 to 249ttgaacggttccact 733 to 747 Cry7Ab LNGST 245 to 249 ttgaacggttccact 733to 747 Cry8Aa LKGTT 256 to 260 ttgaaggtaccact 766 to 780 Cry8Ba LKGSS257 to 261 ttaaaaggctcgagc 769 to 783 Cry8Ca LRGTG 257 to 261ttaagaggaacgggt 769 to 783 Cry9Aa LRQRGTS 252 to 258ctaagacaacgaggcactagt 754 to 774 Cry9Ba1 LRGTS 236 to 240ttacgaggaacgagc 706 to 720 Cry9Ca LRGTN 257 to 261 ttaagaggaacaaat 769to 783 Cry9Da LRGTT 260 to 264 ttaagaggcacaacc 778 to 792 Cry9Ea VRGTN260 to 264 gtagaggaacaaat 778 to 792 Cry10Aa 1RTNT 267 to 271attagaactaatact 799 to 813 Cry16Aa LKLDPN 210 to 215 ttaaaactagatccgaat628 to 645 Cry17Aa IKNKTRDF 224 to 231 ataaaaaataaaactagggatttt 670 to693 Cry19Aa FRTAG 261 to 265 ttagaacagcaggt 781 to 795 Cry19Ba KKQIG 250to 254 aaaaaacaaatagga 748 to 162 Cry20Aa DRSS 245 to 248 gatcgttcaagt733 to 744

[0127] Mutants can be prepared for each of the cry genes mentioned inthis example, based on the models of Examples 1, 2 and 3. The technicalprocedures which can be used to carry out the mutagenesis are similar tothose given in Examples 1, 2 and 3.

Example 5 Overall Increase in the Leucine, Phenylalanine and GlutamicAcid Content of the Cry Proteins

[0128] The overall increase in the leucine, phenylalanine and glutamicacid content of the Cry proteins is described below for the Cry9Ca1toxin. Although this example is carried out on the Cry9Ca1 protein andthe cry9Ca1 gene, its teaching is applicable to all the Cry toxins andall the cry genes. This teaching applies in particular to all the Crytoxins the sequence of which is known and filed in the Genbank database:

www.ncbi.nlm.bih.gov/Genbank/index.html.

[0129] The Genbank accession numbers for the cry genes are available onthe following site:

www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.

[0130] This teaching also applies to all the Cry toxins and cry genes,the sequences of which are not disclosed on Genbank.

[0131] Unlike the strategies described in Examples 1 to 4, the aim isnot to modify a precise region of the toxin so as to integrate aminoacids recognized by pepsin, but to increase, overall, the number ofthese sites by increasing the amount of leucine, of phenylalanine and ofglutamic acid in said toxin. This strategy makes it possible to make theCry toxin more sensitive to pepsin by increasing the percentage ofresidues recognized by pepsin. Glutamic acid (E; Glu) preferentiallysubstitutes for aspartic acid (D; Asp), phenylalanine (F; Phe)preferentially replaces tryptophan (W; Trp) and leucine (L; Leu)preferably replaces valine (V; Val) or isoleucine (I; Ile). Thisstrategy require the creation of a three-dimensional model for theactivated Cry9Ca1 toxin, created from the primary sequence of theprotein by comparison with the three-dimensional structures of Cry1Aa1and Cry3Aa1. The model was created using the Swiss-Model ProteinModelling Server (Peitsch, 1995; Peitsch, 1996; Guex and Peitsch, 1997).The server address is as follows:

www.expasy.ch/swissmod/swiss-model.html.

[0132] Preferably, the substitutions should reach a maximum level of25%. The activated Cry9Ca1 toxin contains 31 aspartic acids, 9tryptophans and 47 valines. There are naturally 26 glutamic acids, 35phenylalanines and 62 leucines. Taking into account a maximumsubstitution of 25% for each of the amino acids, the relative ratios areas follows: Number of residues in native Number of residues in Aminoacid Cry9Ca1 modified Cry9Ca1 Asp (D) 31 24 Glu (E) 26 33 Trp (W)  9  7Phe (F) 35 37 Val (V) 47 36 Leu (L) 61 72

[0133] The substitution of isoleucine (I; Ile) with leucine can also beenvisioned instead of or in addition to the substitution of valine withleucine. There are naturally 27 isoleucines in the Cry9Ca1 toxin. Takinginto account a preferential degree of substitution of 25%, it issufficient to replace 6 isoleucine residues with leucines.

[0134] It is possible to modify the sequence of the cry9Ca1 gene asshown below. The only aim of the demonstration below is to illustratethe example, and it does not in any way limit the scope of theinvention. This demonstration relates to aspartic acid, tryptophan andvaline residue replacement. Those skilled in the art can very easilyadapt this approach to any other cry gene, the sequence of which wouldbe known, and in particular from the sequences available on Genbank andthe accession numbers of which are mentioned on the following site:

www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.

[0135] The cry genes generally expressed in transgenic plants aretruncated genes, i.e. only the gene sequence encoding the activatedtoxin is introduced into these plants. The sequences given in thisexample correspond to this truncated version and extend, depending onwhether it is a case of the gene or the protein, from the initiationcodon or from the first methionine to 15 codons or amino acidsdownstream of the conserved block 5 which limits the activated toxin.

[0136] The sequence of the native and truncated cry9Ca1 gene is given inSEQ ID NO:1.

[0137] The sequence of the native and truncated Cry9Ca1 protein is givenin SEQ ID NO:2.

[0138] The sequence of a modified cry9Ca1 gene in which all the codonsencoding the valine, aspartic acid and tryptophan residues have beenmodified is given in FIG. 1 (SEQ ID NO:9). This modified sequence can beused as a basis for defining the various mutagenesis oligonucleotideswhich may be used. The modified bases are represented in boldcharacters.

[0139] The sequence of a modified Cry9Ca1 protein in which all thevaline, aspartic acid and tryptophan residues have been modified isgiven in FIG. 2 (SEQ ID NO:10) and the modified amino acids arerepresented in bold characters.

[0140] All the mutagenesis oligonucleotides which may make it possibleto perform the valine, phenylalanine and glutamic acid residuereplacements are given in FIG. 3 (SEQ ID NOS:94 to 160). The modifiedbases are represented in bold characters.

[0141] A possibility for the use of certain oligonucleotides to create amodified cry9Ca1 gene in which the replacement with respect to codonsencoding the valine, aspartic acid and tryptophan residues has beencarried out at up to 25% is shown below by way of illustration. The aimof this illustration is to exemplify the strategy developed withoutlimiting the scope of the invention. On the basis of the teaching ofthis example and of FIGS. 1 to 3 (SEQ ID NOS:9 and 10), those skilled inthe art will be able to adapt other combinations of the oligonucleotidesgiven in FIG. 5 (SEQ ID NOS:94 to 160) or other oligonucleotidesprepared along the same principle, in particular for replacingisoleucine residues.

[0142] The sequence of a cry9Ca1 gene modified by replacement withrespect to the codons encoding the valine, aspartic acid and tryptophanresidues up to 25% is given in FIG. 4 (SEQ ID NO:11). The modified basesare in bold.

[0143] The sequence of a Cry9Ca1 protein modified by valine, asparticacid and tryptophan residue replacement up to 25% is given in FIG. 5(SEQ ID NO:12). The modified amino acids are in bold.

[0144] The creation of a modified cry9Ca1 gene in which 25% of thevaline, aspartic acid and tryptophan codons have been modified, and thesequence of which is given in FIG. 4 (SEQ ID NO:11), can be carried outusing, among those given in FIG. 5 (SEQ ID NOS:94 to 160), the followingoligonucleotides:

[0145] Oligonucleotide No. 60

[0146] Oligonucleotide No. 62

[0147] Oligonucleotide No. 67

[0148] Oligonucleotide No. 72

[0149] Oligonucleotide No. 77

[0150] Oligonucleotide No. 78

[0151] Oligonucleotide No. 80

[0152] Oligonucleotide No. 82

[0153] Oligonucleotide No. 83

[0154] Oligonucleotide No. 88

[0155] Oligonucleotide No. 90

[0156] Oligonucleotide No. 92

[0157] Oligonucleotide No. 96

[0158] Oligonucleotide No. 97

[0159] Oligonucleotide No. 103

[0160] Oligonucleotide No. 111

[0161] The method preferably used is a multiple mutagenesis with amixture of the oligonucleotides mentioned immediately above. Thesite-directed mutagenesis procedure is similar to that described inExample 1, the only difference being that a mixture of mutagenesisoligonucleotides is used in this example, whereas a single mutagenesisoligonucleotide is used in Example 1. The protocol used is thatdescribed in Examples 1 to 4. It is common to each of the mutagenesisseries, only the mutagenesis oligonucleotide and the oligonucleotide forinhibition/restoration of the resistance to the antibiotic change.

Example 6 Production of Modified Cry Proteins in B. Thuringiensis andPurification

[0162] The native and modified genes are inserted, with their promoterand terminator sequences, into the E. coli-B. thuringiensis pHT3101shuttle vector (Lereclus et al., 1989).

[0163] The plasmid DNA is prepared by minipreparation according to thealkaline lysis technique (Birboim and Doly, 1979). Each bacterial colonyis grown in 2 ml of LB medium supplemented with the appropriateantibiotic, overnight at 37° C. with shaking (200 rpm). The culture isthen transferred into a microtube and then centrifuged at 13 500 g for 5min. After removal of the supernatant, the bacteria are resuspended in100 μl of a solution of 25 mM Tris-HCl, pH 8, 10 mM EDTA containingRNase A at the final concentration of 100 μg/ml. 200 μl of a solution of0.2 M NaOH, 1% SDS are added and the suspension is mixed twice byinverting the tube. 150 μl of a 2.55 M potassium acetate solution, pH4.5, are added and the suspension is incubated for 5 min in ice. Aftercentrifugation for 15 min at 13 500 g, the supernatant is transferredinto a microtube containing 1 ml of cold ethanol. After centrifugationfor 30 min at 13 500 g, the supernatant is removed and the pellet iswashed with 1 ml of 70% ethanol. The pellet containing the DNA is driedfor a few minutes under vacuum and then taken up in 50 μl of steriledistilled water. The samples are then placed at 65° C. for 30 min.

[0164] The digestions with restriction endonucleases are carried out per1 μg of DNA in a final volume of 20 μl in the presence of one tenth ofthe final volume of 10× buffer recommended by the supplier for eachenzyme and using 5 units of enzyme. The reaction is incubated for 2 to 3h at the optimum temperature for the enzyme.

[0165] Dephosphorylation of the 5′ ends engendered by restriction enzymeis carried out with calf intestine alkaline phosphatase. The reaction iscarried out using 5 μl of 10× dephosphorylation buffer (500 mM Tris-HCl,pH 9.3, 10 mM MgCl2, 1 mM ZnCl₂ and 10 mM spermidine) and one unit ofenzyme per μg of DNA in a final volume of 50 μl. The reaction isincubated for one hour at 37° C. in the case of overhanging 5′ ends orat 55° C. in the case of blunt ends or 3′ overhanging ends. Afterdephosphorylation, the enzyme is then inactivated for 30 min at 65° C.and then removed with two volume for volume extractions with a mixtureof phenol-chloroform-isoamyl alcohol (25-24-1). The ligations arecarried out using T4 phage DNA ligase. They are carried out with anamount of vector equal to 100 ng and an insert/vector molar ratio ofbetween 5 and 10. The final volume of the reaction is 30 μl andcomprises 3 μl of 10× ligase buffer (300 mM Tris-HCl, pH 7.8, 100 mMMgCl2, 100 mM DTT and 10 mM ATP) and 3 units of enzyme. The reaction isincubated overnight at 14° C.

[0166] The construct is inserted into an acrystalliferous strain of B.thuringiensis according to a method derived from that described in 1989by Lereclus et al. and described elsewhere (Rang et al., 1999, 2000). Apreculture of acrystalliferous Bacillus thuringiensis subsp. kurstakiHD-1 is incubated overnight at 37° C. with shaking in 10 ml of BHImedium (Difco). 250 ml of BHI medium are then inoculated with 5 ml ofpreculture and incubated at 37° C. with shaking until the OD at 600 nmof the culture reaches the value of 0.3. The culture is then centrifugedat 1 000 g at 4° C. for 10 min. The supernatant is removed and thebacterial pellet is rinsed with 50 ml of cold sterile distilled water.The bacteria are again centrifuged for 10 min at 1 000 g at 4° C. Thepellet is taken up in 4 ml of a cold, sterile solution of 40% PEG-6000and placed in ice. 200 μl of bacteria are then mixed with 5 μg ofplasmid DNA and then placed in an electroporation cuvette 0.2 cm indiameter. The cuvette is then placed in the electroporation chamber anda current corresponding to the following characteristics: 2.5 kV, 1 000Ω, 25 μF, is supplied. The bacteria are then covered, placed in ice for10 min before being added to 2 ml of BHI medium, and incubated at 37° C.with shaking for 90 min. 200 μl of culture are then plated out ontoPetri dishes containing usual solid medium (IEBC, 1994) supplementedwith erythromycin at a final concentration of 25 μg/ml, and incubatedovernight at 28° C.

[0167] The recombinant strains of Bacillus thuringiensis expressing thenative gene or the mutated genes are cultured in 250 ml of Usual mediumcontaining 25 μg/ml of erythromycin with shaking at 28° C. The bacterialgrowth is verified by observation by phase-contrast light microscopy.The bacteria are grown until bacterial lysis after sporulation. Theculture is then centrifuged at 5 000 g for 10 min. The pellet is washedwith 25 ml of 1 M NaCl and the suspension is again centrifuged at 5 000g for 10 min. The pellet is then taken up in 15 ml of sterile distilledwater containing 1 mM of PMSF, incubated in ice, and treated withultrasound (100 W) for 1 min in order to dissociate the aggregatesbetween the spores and the crystals. The suspension is then loaded ontoa discontinuous NaBr gradient made up of a layer of 4 ml of 38.5%concentration, of 4 layers of 6 ml of 41.9%, 45.3%, 48.9% and 52.7% anda layer of 3 ml of 56.3%. The gradient is then centrifuged at 20 000 gfor 90 min at 20° C. The various components of the suspension (spores,cell debris, parasporal bodies) are positioned in the gradient atvarious levels depending on their density. Each band is recovered andwashed three times with one volume of sterile distilled water. Each bandis observed by phase-contrast light microscopy. The fraction containinginclusion bodies is stored at −20° C. in sterile distilled watercontaining 1 mM of PMSF, for subsequent analysis.

Example 7 Analysis of the Stability of the Proteins to Proteases

[0168] The first stability analysis performed is the verification ofstability to trypsin. The proteins present in the parasporal inclusionbody are solubilized for one hour at 37° C. in solubilizing buffer (50mM Na₂CO₃, pH 10.8, 14.6 mM 2-mercaptoethanol). The suspension is thencentrifuged at 14 000 g for 10 min in order to remove the insolublematerial. One tenth of the total volume of 0.05% trypsin is then addedto the supernatant and the mixture is incubated for 2 h at 37° C. Thecondition of the proteins after trypsin treatment is verified bySDS-polyacrylamide gel analysis according to the Laemmli method (1970).This technique allows the proteins to be separated according to theirmolecular mass by virtue of the presence of SDS, which confers anoverall negative charge on all the proteins. The sample is first treatedby adding one volume of 2× treatment solution (125 mM Tris-HCl, 20%glycerol, 4% SDS, 10% 2-mercaptoethanol, 0.01% bromophenol blue) and isthen denatured for 5 min in boiling water. The sample is then loadedonto the gel and first passes through a first stacking gel made up of a4% acrylamide-bisacrylamide mixture, 0.1% SDS, and 125 mM Tris-HCl, pH6.8. The sample then passes through the separating gel made up of 12%acrylamide-bisacrylamide, 0.1% SDS and 375 mM Tris-HCl, pH 8.8, whichmakes it possible to separate the various proteins as a function oftheir size. The electrophoresis is carried out at 100 V in migrationbuffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1% SDS) until thebromophenol blue leaves the gel. The gel is then stained for one hourwith a solution of 40% methanol-7% acetic acid containing 0.025% ofCoomassie blue and then destained with a 50% methanol-10% acetic acidsolution. The gel is ultimately fixed in a 5% methanol-7% acetic acidsolution.

[0169] The second analysis is the verification of the stability to thedigestive juices of insects. The trypsin-stable toxins are purified byFPLC (Pharmacia) using an anion exchange column (Q-Sepharose)equilibrated with a 40 mM Na₂CO₃ solution, pH 10.7. The elution iscarried out with a gradient of 50 to 500 mM of NaCl. The OD at 280 nm ofthe fractions is measured and the fractions containing the proteins areanalyzed by SDS-polyacrylamide gel electrophoresis. The fractionscontaining the toxin are pooled and dialyzed at 4° C. against distilledwater for approximately 48 h until the proteins precipitate. The proteinsuspension is then centrifuged at 8 000 g and at 4° C. for 30 min. Thetoxins contained in the pellet are resuspended in distilled water andassayed according to Bradford (1976). They are then divided up intoaliquot fractions of 100 μg, lyophilized, and then stored at 4° C.Before they are used, the toxins are solubilized and brought to aconcentration of 10 mg/ml with 25 mM Tris, pH 9.5, for the purpose oftesting their stability to the digestive juices of Ostrinia nubilalislarvae. The digestive juice of the O. nubilalis larvae can be takeneither by regurgitation induced by electric shock according to theprocedure of Ogiwara et al. (1992), or by dissection of the larvae andcollection of the intestinal juice with a pipette according to themethod described by Baines et al. (1994). In both cases, between 100 and200 individuals are required to collect the digestive juice. The juicecollected is centrifuged at 15 000 g for 15 minutes at 4° C. before use.The protein concentration of the digestive juice is determined by theBradford method (BioRad). The reaction is carried out for 15 minutes at37° C. with a 1:1 ratio (based on the protein concentration of thedigestive juice) of toxin to digestive juice. The reaction is stoppedwith a cocktail of protease inhibitors (Protease Inhibitors Set, RocheDiagnostics) mixed with an equivalent volume of 2× treatment solution(125 mM Tris-HCl, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, 0.01%bromo-phenol blue), and then incubated for 5 minutes in boiling water.The proteins are then analyzed by SDS-PAGE according to the proceduredescribed above, in order to determine their resistance to the digestivejuices of the larvae and their possible state of degradation.

[0170] The final type of stability analysis carried out is that ofstability to pepsin. The lyophilized native and modified toxins aredissolved in a gastric buffer (0.5 mg NaCl, 1.75 ml 1M HCl in 250 mlH₂O, pH 2.0) simulating mammalian stomach fluid and containing 0.32% ofpepsin. Samples are removed after 0, 5, 15, 60 and 240 minutes ofincubation at 37° C. and then analyzed by SDS-polyacrylamide gelelectrophoresis as described above. These conditions are identical tothose described in the EPA (United States Environmental ProtectionAgency) No. 4458108.

[0171] This series of analyses makes it possible to visualize the stateof conservation of the native and mutated proteins, and therefore theirstability, to various proteases present in insects (trypsin anddigestive juices) and, consequently, to verify that the mutated proteinshave effectively conserved their stability in insects. These analysesalso make it possible to verify that the mutated proteins areeffectively degraded by pepsin under the conditions similar to thosepresent in the mammalian stomach.

Example 8 Analysis of the Insecticidal Properties

[0172] The analysis of insecticidal properties is carried out throughtwo types of experiment for testing the two steps of the process oftoxicity in insects: receptor site recognition and evaluation of thetoxicity in vivo.

[0173] Analysis of the affinity of the toxins for the receptor site iscarried out using toxin radiolabeled with iodine 125 (¹²⁵I). TheFPLC-purified and lyophilized activated toxins are taken up in storagebuffer (20 mM Tris-HCl, pH 8.6) and analyzed by SDS-PAGE in order toverify their condition. An aliquot fraction is assayed according to theBradford method (1976). The toxins are iodinated according to thechloramine-T method (Markwell, 1982). 25 μμg of toxins are incubated for5 min at ambient temperature with 0.25 mCi of Na-¹²⁵I and an “Iodo-bead”(Pierce) in 50 μl of sodium carbonate buffer (50 mM Na₂CO₃, pH 10). Theiodination reaction is then deposited at the surface of a dextrandesalting column (Pierce) equilibrated with CBS buffer (50 mM Na₂CO₃, pH10.8, 150 mM NaCl) in order to remove the free iodine. The labeling andthe quality of the protein are verified by SDS-PAGE followed byautoradiography. The mean specific activity of a labeled toxin is 100000 cpm/pmol.

[0174] In order to prepare the brush border membrane vesicles (BBMV) onwhich the study of the affinity of the toxins for the receptors iscarried out, the insects are allowed to grow to the final larval stage.The insect used is Ostrinia nubilalis, but the methodology used isapplicable to any other insect species. The use of another insectspecies requires the production conditions and the nutritive medium tobe adapted to each of the species envisioned, which can be readily doneby any individual skilled in the art. The Ostrinia nubilalis larvae areproduced on meridic artificial nutritive medium (Lewis and Lynch, 1969;Reed et al., 1972; Ostlie et al., 1984). The method for producing theOstrinia nubilalis larvae is that described by Huang et al. (1997). Thelarvae are produced individually in 128-well plates (Bio-Ba-128, C-DInternational). Each well contains 2 ml of artificial medium. After tendays, the larvae are transferred into larger dishes (18.4 cm in diameterand 7.6 cm high) containing 300 ml of artificial nutritive medium.Corrugated cardboard is placed inside by way of pupation site. Duringthe larval phase, the temperature of the production cell is 25° C. withconstant light (24 h). The pieces of cardboard containing thechrysalises are transferred into screened cages for the emergence andthe production of the adults. Waxed paper is placed in the case toaccept the eggs. The eggs are removed and kept on hold at 15° C. Theproduction of the adults is carried out at 25° C. with 75% relativehumidity and a photoperiod of 14 h.

[0175] To carry out the tests of affinity of the toxins for the receptorsites, the larvae are collected at the beginning of the 5th larval stageand placed under fasting conditions for 6 hours. They are then removedand placed on ice for 5 minutes. The larvae are dissected and thedigestive tube is removed. The dissected digestive tubes are pooled ingroups of 20, placed in a cryotube containing MET buffer (300 mMmannitol, 5 mM EGTA, 17 mM Tris-HCl, pH 7.5), frozen in liquid nitrogenand stored at −80° C.

[0176] The BBMVs are prepared according to the differential magnesiumprecipitation method (Wolfersberger et al., 1987; Nielsen-LeRoux andCharles, 1992). The BBMVs are taken up in TBS buffer (20 mM Tris-HCl, pH8.5, 150 mM NaCl) and the total protein concentration is determined bythe Bradford method using the Biorad kit and bovine serum albumin (BSA)as standard (Bradford, 1976).

[0177] The in vitro receptor recognition assays are carried out in 1.5ml polyethylene microtubes, in 20 mM sodium phosphate buffer, pH 7.4,containing 0.15 M of NaCl and 0.1% of bovine serum albumin (PBS/BSA).The assays are carried out, in duplicate, at ambient temperature in atotal volume of 100 μl, with 10 μg of BBMV protein. The toxins attachedto the BBMVs are separated from the free toxins by centrifugation at 14000 g for 10 min at ambient temperature. The pellets of each sample,containing the toxin attached to the membrane, are rinsed twice with 200μl of cold PBS/BSA buffer (20 mM Tris/HCl, 150 mM NaCl, 0.1% BSA, pH8.5) and then centrifuged. The pellets are finally resuspended in 200 μlof PBS/BSA buffer and added to 3 ml of HiSafe 3 scintillant cocktail(Pharmacia) in a scintillation vial. The counting is performed in aliquid scintillation counter.

[0178] The direct binding assays are carried out according to theNielsen-LeRoux and Charles protocol (1992). 30 μg of BBMV per microtubeare incubated with a series of concentrations of 1 to 100 mM of toxinlabeled with ¹²⁵I-iodine in Tris/BSA buffer (20 mM Tris/HCl, 150 mMNaCl, 0.1% BSA, pH 8.5). The amount of nonspecific attachment isdetermined in parallel experiments in the presence of a 300-fold excessof unlabeled toxin. After incubation for 90 minutes at ambienttemperature, the samples are centrifuged at 14 000 g for 10 minutes at4° C. The pellets are rinsed twice with cold Tris/BSA buffer andresuspended in 150 μl of the same buffer and added to 3 ml of HiSafe 3scintillant cocktail (Pharmacia) in a scintillation vial. Eachexperiment is carried out in duplicate and each experimental point iscounted twice in a liquid scintillation counter. The data are analyzedusing the LIGAND software (Munson and Rodbard, 1980) marketed by thecompany Biosoft.

[0179] The homologous competition experiments are carried out asdescribed above for the direct binding experiments, with 10 μg of BBMVsin a total volume of 100 μl for 90 min at ambient temperature. The BBMVsare incubated in a fixed concentration of 10 nM of toxin labeled with125I-iodine in the presence of a series of concentrations (from 0.1 to300 times the concentration of the labeled toxin) in Tris/BSA buffer.The value for the nonspecific binding (the binding always present in thepresence of a 300-fold excess of the unlabeled toxin) is subtracted fromthe total value counted. Each experiment is carried out in duplicate andeach experimental point is counted twice in a liquid scintillationcounter. The data are analyzed using the LIGAND software (Munson andRodbard, 1980) marketed by the company Biosoft.

[0180] The in vivo toxicity assays are carried out according to theprocedure described by Lambert et al. (1996). The activated andsolubilized toxin is incorporated into the nutritive medium at variousconcentrations either side of the 50% lethal dose (LD50) of Cry9Ca1 forOstrinia nubilalis, which is 96.6 ng of toxin per cm² of surface area ofmedium. Six doses, of 0.1 ng/cm², 1 ng/cm², 10 ng/cm², 100 ng/cm², 1 000ng/cm² and 10 000 ng/cm² evaluate the LD50 values of the native andmodified toxins. The toxicity assays are carried out on neonatal larvaein plates containing 24 wells of 2 cm² (Multiwell-24 plates, ComingCostar Corp.). 50 μl of each of the dilutions of toxin are plated outonto the medium and dried under a flow hood. One larva is placed in eachwell and a total of 24 larvae is used for each dose (one plate perdose). For each dose the assay is repeated at least three times. Acontrol is carried out with distilled water. The plates are covered andplaced at 25° C., 70% relative humidity and with a photoperiod of 16 h.The mortality is controlled after 7 days and the LD50 is calculatedaccording to the probit method (Finney, 1971).

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1 160 1 2019 DNA Bacillus thuringiensis CDS (1)..(2019) 1 atg aat cgaaat aat caa aat gaa tat gaa att att gat gcc ccc cat 48 Met Asn Arg AsnAsn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 tgt ggg tgtcca tca gat gac gat gtg agg tat cct ttg gca agt gac 96 Cys Gly Cys ProSer Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 cca aat gca gcgtta caa aat atg aac tat aaa gat tac tta caa atg 144 Pro Asn Ala Ala LeuGln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 aca gat gag gac tacact gat tct tat ata aat cct agt tta tct att 192 Thr Asp Glu Asp Tyr ThrAsp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 agt ggt aga gat gca gttcag act gcg ctt act gtt gtt ggg aga ata 240 Ser Gly Arg Asp Ala Val GlnThr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 ctc ggg gct tta ggt gttccg ttt tct gga caa ata gtg agt ttt tat 288 Leu Gly Ala Leu Gly Val ProPhe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 caa ttc ctt tta aat aca ctgtgg cca gtt aat gat aca gct ata tgg 336 Gln Phe Leu Leu Asn Thr Leu TrpPro Val Asn Asp Thr Ala Ile Trp 100 105 110 gaa gct ttc atg cga cag gtggag gaa ctt gtc aat caa caa ata aca 384 Glu Ala Phe Met Arg Gln Val GluGlu Leu Val Asn Gln Gln Ile Thr 115 120 125 gaa ttt gca aga aat cag gcactt gca aga ttg caa gga tta gga gac 432 Glu Phe Ala Arg Asn Gln Ala LeuAla Arg Leu Gln Gly Leu Gly Asp 130 135 140 tct ttt aat gta tat caa cgttcc ctt caa aat tgg ttg gct gat cga 480 Ser Phe Asn Val Tyr Gln Arg SerLeu Gln Asn Trp Leu Ala Asp Arg 145 150 155 160 aat gat aca cga aat ttaagt gtt gtt cgt gct caa ttt ata gct tta 528 Asn Asp Thr Arg Asn Leu SerVal Val Arg Ala Gln Phe Ile Ala Leu 165 170 175 gac ctt gat ttt gtt aatgct att cca ttg ttt gca gta aat gga cag 576 Asp Leu Asp Phe Val Asn AlaIle Pro Leu Phe Ala Val Asn Gly Gln 180 185 190 cag gtt cca tta ctg tcagta tat gca caa gct gtg aat tta cat ttg 624 Gln Val Pro Leu Leu Ser ValTyr Ala Gln Ala Val Asn Leu His Leu 195 200 205 tta tta tta aaa gat gcatct ctt ttt gga gaa gga tgg gga ttc aca 672 Leu Leu Leu Lys Asp Ala SerLeu Phe Gly Glu Gly Trp Gly Phe Thr 210 215 220 cag ggg gaa att tcc acatat tat gac cgt caa ttg gaa cta acc gct 720 Gln Gly Glu Ile Ser Thr TyrTyr Asp Arg Gln Leu Glu Leu Thr Ala 225 230 235 240 aag tac act aat tactgt gaa act tgg tat aat aca ggt tta gat cgt 768 Lys Tyr Thr Asn Tyr CysGlu Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250 255 tta aga gga aca aatact gaa agt tgg tta aga tat cat caa ttc cgt 816 Leu Arg Gly Thr Asn ThrGlu Ser Trp Leu Arg Tyr His Gln Phe Arg 260 265 270 aga gaa atg act ttagtg gta tta gat gtt gtg gcg cta ttt cca tat 864 Arg Glu Met Thr Leu ValVal Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 tat gat gta cga ctttat cca acg gga tca aac cca cag ctt aca cgt 912 Tyr Asp Val Arg Leu TyrPro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 gag gta tat aca gatccg att gta ttt aat cca cca gct aat gtt gga 960 Glu Val Tyr Thr Asp ProIle Val Phe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 ctt tgc cga cgttgg ggt act aat ccc tat aat act ttt tct gag ctc 1008 Leu Cys Arg Arg TrpGly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 gaa aat gcc ttcatt cgc cca cca cat ctt ttt gat agg ctg aat agc 1056 Glu Asn Ala Phe IleArg Pro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 tta aca atc agcagt aat cga ttt cca gtt tca tct aat ttt atg gat 1104 Leu Thr Ile Ser SerAsn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 tat tgg tca ggacat acg tta cgc cgt agt tat ctg aac gat tca gca 1152 Tyr Trp Ser Gly HisThr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 gta caa gaa gatagt tat ggc cta att aca acc aca aga gca aca att 1200 Val Gln Glu Asp SerTyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395 400 aat ccc ggagtt gat gga aca aac cgc ata gag tca acg gca gta gat 1248 Asn Pro Gly ValAsp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415 ttt cgt tctgca ttg ata ggt ata tat ggc gtg aat aga gct tct ttt 1296 Phe Arg Ser AlaLeu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe 420 425 430 gtc cca ggaggc ttg ttt aat ggt acg act tct cct gct aat gga gga 1344 Val Pro Gly GlyLeu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435 440 445 tgt aga gatctc tat gat aca aat gat gaa tta cca cca gat gaa agt 1392 Cys Arg Asp LeuTyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser 450 455 460 acc gga agttca acc cat aga cta tct cat gtt acc ttt ttt agc ttt 1440 Thr Gly Ser SerThr His Arg Leu Ser His Val Thr Phe Phe Ser Phe 465 470 475 480 caa actaat cag gct gga tct ata gct aat gca gga agt gta cct act 1488 Gln Thr AsnGln Ala Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490 495 tat gtttgg acc cgt cgt gat gtg gac ctt aat aat acg att acc cca 1536 Tyr Val TrpThr Arg Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505 510 aat agaatt aca caa tta cca ttg gta aag gca tct gca cct gtt tcg 1584 Asn Arg IleThr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 ggt actacg gtc tta aaa ggt cca gga ttt aca gga ggg ggt ata ctc 1632 Gly Thr ThrVal Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 cga agaaca act aat ggc aca ttt gga acg tta aga gta acg gtt aat 1680 Arg Arg ThrThr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560 tcacca tta aca caa caa tat cgc cta aga gtt cgt ttt gcc tca aca 1728 Ser ProLeu Thr Gln Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 ggaaat ttc agt ata agg gta ctc cgt gga ggg gtt tct atc ggt gat 1776 Gly AsnPhe Ser Ile Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 gttaga tta ggg agc aca atg aac aga ggg cag gaa cta act tac gaa 1824 Val ArgLeu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 tccttt ttc aca aga gag ttt act act act ggt ccg ttc aat ccg cct 1872 Ser PhePhe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615 620 tttaca ttt aca caa gct caa gag att cta aca gtg aat gca gaa ggt 1920 Phe ThrPhe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625 630 635 640gtt agc acc ggt ggt gaa tat tat ata gat aga att gaa att gtc cct 1968 ValSer Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655gtg aat ccg gca cga gaa gcg gaa gag gat tta gaa gcg gcg aag aaa 2016 ValAsn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys 660 665 670gcg 2019 Ala 2 673 PRT Bacillus thuringiensis 2 Met Asn Arg Asn Asn GlnAsn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 Cys Gly Cys Pro SerAsp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 Pro Asn Ala Ala LeuGln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 Thr Asp Glu Asp TyrThr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 Ser Gly Arg Asp AlaVal Gln Thr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 Leu Gly Ala LeuGly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 Gln Phe Leu LeuAsn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp 100 105 110 Glu Ala PheMet Arg Gln Val Glu Glu Leu Val Asn Gln Gln Ile Thr 115 120 125 Glu PheAla Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135 140 SerPhe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg 145 150 155160 Asn Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu 165170 175 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln180 185 190 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn Leu HisLeu 195 200 205 Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly Trp GlyPhe Thr 210 215 220 Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu GluLeu Thr Ala 225 230 235 240 Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr AsnThr Gly Leu Asp Arg 245 250 255 Leu Arg Gly Thr Asn Thr Glu Ser Trp LeuArg Tyr His Gln Phe Arg 260 265 270 Arg Glu Met Thr Leu Val Val Leu AspVal Val Ala Leu Phe Pro Tyr 275 280 285 Tyr Asp Val Arg Leu Tyr Pro ThrGly Ser Asn Pro Gln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro IleVal Phe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 Leu Cys Arg Arg TrpGly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 Glu Asn Ala PheIle Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 Leu Thr IleSer Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 Tyr TrpSer Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 ValGln Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395400 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405410 415 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe420 425 430 Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn GlyGly 435 440 445 Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro Pro AspGlu Ser 450 455 460 Thr Gly Ser Ser Thr His Arg Leu Ser His Val Thr PhePhe Ser Phe 465 470 475 480 Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn AlaGly Ser Val Pro Thr 485 490 495 Tyr Val Trp Thr Arg Arg Asp Val Asp LeuAsn Asn Thr Ile Thr Pro 500 505 510 Asn Arg Ile Thr Gln Leu Pro Leu ValLys Ala Ser Ala Pro Val Ser 515 520 525 Gly Thr Thr Val Leu Lys Gly ProGly Phe Thr Gly Gly Gly Ile Leu 530 535 540 Arg Arg Thr Thr Asn Gly ThrPhe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560 Ser Pro Leu Thr GlnGln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 Gly Asn Phe SerIle Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 Val Arg LeuGly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 Ser PhePhe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615 620 PheThr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625 630 635640 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645650 655 Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys660 665 670 Ala 3 2019 DNA Artificial sequence Artificial sequencedescription Cry9Ca1 Leu- 164 3 atg aat cga aat aat caa aat gaa tat gaaatt att gat gcc ccc cat 48 Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu IleIle Asp Ala Pro His 1 5 10 15 tgt ggg tgt cca tca gat gac gat gtg aggtat cct ttg gca agt gac 96 Cys Gly Cys Pro Ser Asp Asp Asp Val Arg TyrPro Leu Ala Ser Asp 20 25 30 cca aat gca gcg tta caa aat atg aac tat aaagat tac tta caa atg 144 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys AspTyr Leu Gln Met 35 40 45 aca gat gag gac tac act gat tct tat ata aat cctagt tta tct att 192 Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro SerLeu Ser Ile 50 55 60 agt ggt aga gat gca gtt cag act gcg ctt act gtt gttggg aga ata 240 Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val Val GlyArg Ile 65 70 75 80 ctc ggg gct tta ggt gtt ccg ttt tct gga caa ata gtgagt ttt tat 288 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Val SerPhe Tyr 85 90 95 caa ttc ctt tta aat aca ctg tgg cca gtt aat gat aca gctata tgg 336 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala IleTrp 100 105 110 gaa gct ttc atg cga cag gtg gag gaa ctt gtc aat caa caaata aca 384 Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln IleThr 115 120 125 gaa ttt gca aga aat cag gca ctt gca aga ttg caa gga ttagga gac 432 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu GlyAsp 130 135 140 tct ttt aat gta tat caa cgt tcc ctt caa aat tgg ttg gctgat cga 480 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala AspArg 145 150 155 160 aat gat aca tta aat tta agt gtt gtt cgt gct caa tttata gct tta 528 Asn Asp Thr Leu Asn Leu Ser Val Val Arg Ala Gln Phe IleAla Leu 165 170 175 gac ctt gat ttt gtt aat gct att cca ttg ttt gca gtaaat gga cag 576 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val AsnGly Gln 180 185 190 cag gtt cca tta ctg tca gta tat gca caa gct gtg aattta cat ttg 624 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn LeuHis Leu 195 200 205 tta tta tta aaa gat gca tct ctt ttt gga gaa gga tgggga ttc aca 672 Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly Trp GlyPhe Thr 210 215 220 cag ggg gaa att tcc aca tat tat gac cgt caa ttg gaacta acc gct 720 Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu Glu LeuThr Ala 225 230 235 240 aag tac act aat tac tgt gaa act tgg tat aat acaggt tta gat cgt 768 Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr GlyLeu Asp Arg 245 250 255 tta aga gga aca aat act gaa agt tgg tta aga tatcat caa ttc cgt 816 Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg Tyr HisGln Phe Arg 260 265 270 aga gaa atg act tta gtg gta tta gat gtt gtg gcgcta ttt cca tat 864 Arg Glu Met Thr Leu Val Val Leu Asp Val Val Ala LeuPhe Pro Tyr 275 280 285 tat gat gta cga ctt tat cca acg gga tca aac ccacag ctt aca cgt 912 Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn Pro GlnLeu Thr Arg 290 295 300 gag gta tat aca gat ccg att gta ttt aat cca ccagct aat gtt gga 960 Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro AlaAsn Val Gly 305 310 315 320 ctt tgc cga cgt tgg ggt act aat ccc tat aatact ttt tct gag ctc 1008 Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn ThrPhe Ser Glu Leu 325 330 335 gaa aat gcc ttc att cgc cca cca cat ctt tttgat agg ctg aat agc 1056 Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe AspArg Leu Asn Ser 340 345 350 tta aca atc agc agt aat cga ttt cca gtt tcatct aat ttt atg gat 1104 Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Ser SerAsn Phe Met Asp 355 360 365 tat tgg tca gga cat acg tta cgc cgt agt tatctg aac gat tca gca 1152 Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Tyr LeuAsn Asp Ser Ala 370 375 380 gta caa gaa gat agt tat ggc cta att aca accaca aga gca aca att 1200 Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Thr ThrArg Ala Thr Ile 385 390 395 400 aat ccc gga gtt gat gga aca aac cgc atagag tca acg gca gta gat 1248 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile GluSer Thr Ala Val Asp 405 410 415 ttt cgt tct gca ttg ata ggt ata tat ggcgtg aat aga gct tct ttt 1296 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly ValAsn Arg Ala Ser Phe 420 425 430 gtc cca gga ggc ttg ttt aat ggt acg acttct cct gct aat gga gga 1344 Val Pro Gly Gly Leu Phe Asn Gly Thr Thr SerPro Ala Asn Gly Gly 435 440 445 tgt aga gat ctc tat gat aca aat gat gaatta cca cca gat gaa agt 1392 Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu LeuPro Pro Asp Glu Ser 450 455 460 acc gga agt tca acc cat aga cta tct catgtt acc ttt ttt agc ttt 1440 Thr Gly Ser Ser Thr His Arg Leu Ser His ValThr Phe Phe Ser Phe 465 470 475 480 caa act aat cag gct gga tct ata gctaat gca gga agt gta cct act 1488 Gln Thr Asn Gln Ala Gly Ser Ile Ala AsnAla Gly Ser Val Pro Thr 485 490 495 tat gtt tgg acc cgt cgt gat gtg gacctt aat aat acg att acc cca 1536 Tyr Val Trp Thr Arg Arg Asp Val Asp LeuAsn Asn Thr Ile Thr Pro 500 505 510 aat aga att aca caa tta cca ttg gtaaag gca tct gca cct gtt tcg 1584 Asn Arg Ile Thr Gln Leu Pro Leu Val LysAla Ser Ala Pro Val Ser 515 520 525 ggt act acg gtc tta aaa ggt cca ggattt aca gga ggg ggt ata ctc 1632 Gly Thr Thr Val Leu Lys Gly Pro Gly PheThr Gly Gly Gly Ile Leu 530 535 540 cga aga aca act aat ggc aca ttt ggaacg tta aga gta acg gtt aat 1680 Arg Arg Thr Thr Asn Gly Thr Phe Gly ThrLeu Arg Val Thr Val Asn 545 550 555 560 tca cca tta aca caa caa tat cgccta aga gtt cgt ttt gcc tca aca 1728 Ser Pro Leu Thr Gln Gln Tyr Arg LeuArg Val Arg Phe Ala Ser Thr 565 570 575 gga aat ttc agt ata agg gta ctccgt gga ggg gtt tct atc ggt gat 1776 Gly Asn Phe Ser Ile Arg Val Leu ArgGly Gly Val Ser Ile Gly Asp 580 585 590 gtt aga tta ggg agc aca atg aacaga ggg cag gaa cta act tac gaa 1824 Val Arg Leu Gly Ser Thr Met Asn ArgGly Gln Glu Leu Thr Tyr Glu 595 600 605 tcc ttt ttc aca aga gag ttt actact act ggt ccg ttc aat ccg cct 1872 Ser Phe Phe Thr Arg Glu Phe Thr ThrThr Gly Pro Phe Asn Pro Pro 610 615 620 ttt aca ttt aca caa gct caa gagatt cta aca gtg aat gca gaa ggt 1920 Phe Thr Phe Thr Gln Ala Gln Glu IleLeu Thr Val Asn Ala Glu Gly 625 630 635 640 gtt agc acc ggt ggt gaa tattat ata gat aga att gaa att gtc cct 1968 Val Ser Thr Gly Gly Glu Tyr TyrIle Asp Arg Ile Glu Ile Val Pro 645 650 655 gtg aat ccg gca cga gaa gcggaa gag gat tta gaa gcg gcg aag aaa 2016 Val Asn Pro Ala Arg Glu Ala GluGlu Asp Leu Glu Ala Ala Lys Lys 660 665 670 gcg 2019 Ala 4 673 PRTArtificial sequence Artificial sequence description Cry9Ca1 Leu- 164 4Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 1015 Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 2530 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 4045 Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 5560 Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val Val Gly Arg Ile 65 7075 80 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr 8590 95 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp100 105 110 Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln IleThr 115 120 125 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly LeuGly Asp 130 135 140 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp LeuAla Asp Arg 145 150 155 160 Asn Asp Thr Leu Asn Leu Ser Val Val Arg AlaGln Phe Ile Ala Leu 165 170 175 Asp Leu Asp Phe Val Asn Ala Ile Pro LeuPhe Ala Val Asn Gly Gln 180 185 190 Gln Val Pro Leu Leu Ser Val Tyr AlaGln Ala Val Asn Leu His Leu 195 200 205 Leu Leu Leu Lys Asp Ala Ser LeuPhe Gly Glu Gly Trp Gly Phe Thr 210 215 220 Gln Gly Glu Ile Ser Thr TyrTyr Asp Arg Gln Leu Glu Leu Thr Ala 225 230 235 240 Lys Tyr Thr Asn TyrCys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250 255 Leu Arg Gly ThrAsn Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Arg 260 265 270 Arg Glu MetThr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 Tyr AspVal Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 GluVal Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gly 305 310 315320 Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325330 335 Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser340 345 350 Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe MetAsp 355 360 365 Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn AspSer Ala 370 375 380 Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr ArgAla Thr Ile 385 390 395 400 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile GluSer Thr Ala Val Asp 405 410 415 Phe Arg Ser Ala Leu Ile Gly Ile Tyr GlyVal Asn Arg Ala Ser Phe 420 425 430 Val Pro Gly Gly Leu Phe Asn Gly ThrThr Ser Pro Ala Asn Gly Gly 435 440 445 Cys Arg Asp Leu Tyr Asp Thr AsnAsp Glu Leu Pro Pro Asp Glu Ser 450 455 460 Thr Gly Ser Ser Thr His ArgLeu Ser His Val Thr Phe Phe Ser Phe 465 470 475 480 Gln Thr Asn Gln AlaGly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490 495 Tyr Val Trp ThrArg Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505 510 Asn Arg IleThr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 Gly ThrThr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 ArgArg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555560 Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565570 575 Gly Asn Phe Ser Ile Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp580 585 590 Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr TyrGlu 595 600 605 Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe AsnPro Pro 610 615 620 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val AsnAla Glu Gly 625 630 635 640 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp ArgIle Glu Ile Val Pro 645 650 655 Val Asn Pro Ala Arg Glu Ala Glu Glu AspLeu Glu Ala Ala Lys Lys 660 665 670 Ala 5 2019 DNA Artificial sequenceArtificial sequence description Cry9Ca1 Phe- 164 5 atg aat cga aat aatcaa aat gaa tat gaa att att gat gcc ccc cat 48 Met Asn Arg Asn Asn GlnAsn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 tgt ggg tgt cca tcagat gac gat gtg agg tat cct ttg gca agt gac 96 Cys Gly Cys Pro Ser AspAsp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 cca aat gca gcg tta caaaat atg aac tat aaa gat tac tta caa atg 144 Pro Asn Ala Ala Leu Gln AsnMet Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 aca gat gag gac tac act gattct tat ata aat cct agt tta tct att 192 Thr Asp Glu Asp Tyr Thr Asp SerTyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 agt ggt aga gat gca gtt cag actgcg ctt act gtt gtt ggg aga ata 240 Ser Gly Arg Asp Ala Val Gln Thr AlaLeu Thr Val Val Gly Arg Ile 65 70 75 80 ctc ggg gct tta ggt gtt ccg ttttct gga caa ata gtg agt ttt tat 288 Leu Gly Ala Leu Gly Val Pro Phe SerGly Gln Ile Val Ser Phe Tyr 85 90 95 caa ttc ctt tta aat aca ctg tgg ccagtt aat gat aca gct ata tgg 336 Gln Phe Leu Leu Asn Thr Leu Trp Pro ValAsn Asp Thr Ala Ile Trp 100 105 110 gaa gct ttc atg cga cag gtg gag gaactt gtc aat caa caa ata aca 384 Glu Ala Phe Met Arg Gln Val Glu Glu LeuVal Asn Gln Gln Ile Thr 115 120 125 gaa ttt gca aga aat cag gca ctt gcaaga ttg caa gga tta gga gac 432 Glu Phe Ala Arg Asn Gln Ala Leu Ala ArgLeu Gln Gly Leu Gly Asp 130 135 140 tct ttt aat gta tat caa cgt tcc cttcaa aat tgg ttg gct gat cga 480 Ser Phe Asn Val Tyr Gln Arg Ser Leu GlnAsn Trp Leu Ala Asp Arg 145 150 155 160 aat gat aca ttt aat tta agt gttgtt cgt gct caa ttt ata gct tta 528 Asn Asp Thr Phe Asn Leu Ser Val ValArg Ala Gln Phe Ile Ala Leu 165 170 175 gac ctt gat ttt gtt aat gct attcca ttg ttt gca gta aat gga cag 576 Asp Leu Asp Phe Val Asn Ala Ile ProLeu Phe Ala Val Asn Gly Gln 180 185 190 cag gtt cca tta ctg tca gta tatgca caa gct gtg aat tta cat ttg 624 Gln Val Pro Leu Leu Ser Val Tyr AlaGln Ala Val Asn Leu His Leu 195 200 205 tta tta tta aaa gat gca tct cttttt gga gaa gga tgg gga ttc aca 672 Leu Leu Leu Lys Asp Ala Ser Leu PheGly Glu Gly Trp Gly Phe Thr 210 215 220 cag ggg gaa att tcc aca tat tatgac cgt caa ttg gaa cta acc gct 720 Gln Gly Glu Ile Ser Thr Tyr Tyr AspArg Gln Leu Glu Leu Thr Ala 225 230 235 240 aag tac act aat tac tgt gaaact tgg tat aat aca ggt tta gat cgt 768 Lys Tyr Thr Asn Tyr Cys Glu ThrTrp Tyr Asn Thr Gly Leu Asp Arg 245 250 255 tta aga gga aca aat act gaaagt tgg tta aga tat cat caa ttc cgt 816 Leu Arg Gly Thr Asn Thr Glu SerTrp Leu Arg Tyr His Gln Phe Arg 260 265 270 aga gaa atg act tta gtg gtatta gat gtt gtg gcg cta ttt cca tat 864 Arg Glu Met Thr Leu Val Val LeuAsp Val Val Ala Leu Phe Pro Tyr 275 280 285 tat gat gta cga ctt tat ccaacg gga tca aac cca cag ctt aca cgt 912 Tyr Asp Val Arg Leu Tyr Pro ThrGly Ser Asn Pro Gln Leu Thr Arg 290 295 300 gag gta tat aca gat ccg attgta ttt aat cca cca gct aat gtt gga 960 Glu Val Tyr Thr Asp Pro Ile ValPhe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 ctt tgc cga cgt tgg ggtact aat ccc tat aat act ttt tct gag ctc 1008 Leu Cys Arg Arg Trp Gly ThrAsn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 gaa aat gcc ttc att cgccca cca cat ctt ttt gat agg ctg aat agc 1056 Glu Asn Ala Phe Ile Arg ProPro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 tta aca atc agc agt aatcga ttt cca gtt tca tct aat ttt atg gat 1104 Leu Thr Ile Ser Ser Asn ArgPhe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 tat tgg tca gga cat acgtta cgc cgt agt tat ctg aac gat tca gca 1152 Tyr Trp Ser Gly His Thr LeuArg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 gta caa gaa gat agt tatggc cta att aca acc aca aga gca aca att 1200 Val Gln Glu Asp Ser Tyr GlyLeu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395 400 aat ccc gga gtt gatgga aca aac cgc ata gag tca acg gca gta gat 1248 Asn Pro Gly Val Asp GlyThr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415 ttt cgt tct gca ttgata ggt ata tat ggc gtg aat aga gct tct ttt 1296 Phe Arg Ser Ala Leu IleGly Ile Tyr Gly Val Asn Arg Ala Ser Phe 420 425 430 gtc cca gga ggc ttgttt aat ggt acg act tct cct gct aat gga gga 1344 Val Pro Gly Gly Leu PheAsn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435 440 445 tgt aga gat ctc tatgat aca aat gat gaa tta cca cca gat gaa agt 1392 Cys Arg Asp Leu Tyr AspThr Asn Asp Glu Leu Pro Pro Asp Glu Ser 450 455 460 acc gga agt tca acccat aga cta tct cat gtt acc ttt ttt agc ttt 1440 Thr Gly Ser Ser Thr HisArg Leu Ser His Val Thr Phe Phe Ser Phe 465 470 475 480 caa act aat caggct gga tct ata gct aat gca gga agt gta cct act 1488 Gln Thr Asn Gln AlaGly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490 495 tat gtt tgg acccgt cgt gat gtg gac ctt aat aat acg att acc cca 1536 Tyr Val Trp Thr ArgArg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505 510 aat aga att acacaa tta cca ttg gta aag gca tct gca cct gtt tcg 1584 Asn Arg Ile Thr GlnLeu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 ggt act acg gtctta aaa ggt cca gga ttt aca gga ggg ggt ata ctc 1632 Gly Thr Thr Val LeuLys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 cga aga aca actaat ggc aca ttt gga acg tta aga gta acg gtt aat 1680 Arg Arg Thr Thr AsnGly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560 tca cca ttaaca caa caa tat cgc cta aga gtt cgt ttt gcc tca aca 1728 Ser Pro Leu ThrGln Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 gga aat ttcagt ata agg gta ctc cgt gga ggg gtt tct atc ggt gat 1776 Gly Asn Phe SerIle Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 gtt aga ttaggg agc aca atg aac aga ggg cag gaa cta act tac gaa 1824 Val Arg Leu GlySer Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 tcc ttt ttcaca aga gag ttt act act act ggt ccg ttc aat ccg cct 1872 Ser Phe Phe ThrArg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615 620 ttt aca tttaca caa gct caa gag att cta aca gtg aat gca gaa ggt 1920 Phe Thr Phe ThrGln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625 630 635 640 gtt agcacc ggt ggt gaa tat tat ata gat aga att gaa att gtc cct 1968 Val Ser ThrGly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655 gtg aatccg gca cga gaa gcg gaa gag gat tta gaa gcg gcg aag aaa 2016 Val Asn ProAla Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys 660 665 670 gcg 2019Ala 6 673 PRT Artificial sequence Artificial sequence descriptionCry9Ca1 Phe- 164 6 Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile AspAla Pro His 1 5 10 15 Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr ProLeu Ala Ser Asp 20 25 30 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys AspTyr Leu Gln Met 35 40 45 Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn ProSer Leu Ser Ile 50 55 60 Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr ValVal Gly Arg Ile 65 70 75 80 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly GlnIle Val Ser Phe Tyr 85 90 95 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val AsnAsp Thr Ala Ile Trp 100 105 110 Glu Ala Phe Met Arg Gln Val Glu Glu LeuVal Asn Gln Gln Ile Thr 115 120 125 Glu Phe Ala Arg Asn Gln Ala Leu AlaArg Leu Gln Gly Leu Gly Asp 130 135 140 Ser Phe Asn Val Tyr Gln Arg SerLeu Gln Asn Trp Leu Ala Asp Arg 145 150 155 160 Asn Asp Thr Phe Asn LeuSer Val Val Arg Ala Gln Phe Ile Ala Leu 165 170 175 Asp Leu Asp Phe ValAsn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln 180 185 190 Gln Val Pro LeuLeu Ser Val Tyr Ala Gln Ala Val Asn Leu His Leu 195 200 205 Leu Leu LeuLys Asp Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Thr 210 215 220 Gln GlyGlu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Ala 225 230 235 240Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250255 Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Arg 260265 270 Arg Glu Met Thr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr275 280 285 Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu ThrArg 290 295 300 Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala AsnVal Gly 305 310 315 320 Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn ThrPhe Ser Glu Leu 325 330 335 Glu Asn Ala Phe Ile Arg Pro Pro His Leu PheAsp Arg Leu Asn Ser 340 345 350 Leu Thr Ile Ser Ser Asn Arg Phe Pro ValSer Ser Asn Phe Met Asp 355 360 365 Tyr Trp Ser Gly His Thr Leu Arg ArgSer Tyr Leu Asn Asp Ser Ala 370 375 380 Val Gln Glu Asp Ser Tyr Gly LeuIle Thr Thr Thr Arg Ala Thr Ile 385 390 395 400 Asn Pro Gly Val Asp GlyThr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415 Phe Arg Ser Ala LeuIle Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe 420 425 430 Val Pro Gly GlyLeu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435 440 445 Cys Arg AspLeu Tyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser 450 455 460 Thr GlySer Ser Thr His Arg Leu Ser His Val Thr Phe Phe Ser Phe 465 470 475 480Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490495 Tyr Val Trp Thr Arg Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500505 510 Asn Arg Ile Thr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser515 520 525 Gly Thr Thr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly IleLeu 530 535 540 Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val ThrVal Asn 545 550 555 560 Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Val ArgPhe Ala Ser Thr 565 570 575 Gly Asn Phe Ser Ile Arg Val Leu Arg Gly GlyVal Ser Ile Gly Asp 580 585 590 Val Arg Leu Gly Ser Thr Met Asn Arg GlyGln Glu Leu Thr Tyr Glu 595 600 605 Ser Phe Phe Thr Arg Glu Phe Thr ThrThr Gly Pro Phe Asn Pro Pro 610 615 620 Phe Thr Phe Thr Gln Ala Gln GluIle Leu Thr Val Asn Ala Glu Gly 625 630 635 640 Val Ser Thr Gly Gly GluTyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655 Val Asn Pro Ala ArgGlu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys 660 665 670 Ala 7 2019 DNAArtificial sequence Artificial sequence description Cry9Ca1 Glu- 164 7atg aat cga aat aat caa aat gaa tat gaa att att gat gcc ccc cat 48 MetAsn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15tgt ggg tgt cca tca gat gac gat gtg agg tat cct ttg gca agt gac 96 CysGly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 ccaaat gca gcg tta caa aat atg aac tat aaa gat tac tta caa atg 144 Pro AsnAla Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 aca gatgag gac tac act gat tct tat ata aat cct agt tta tct att 192 Thr Asp GluAsp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 agt ggt agagat gca gtt cag act gcg ctt act gtt gtt ggg aga ata 240 Ser Gly Arg AspAla Val Gln Thr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 ctc ggg gcttta ggt gtt ccg ttt tct gga caa ata gtg agt ttt tat 288 Leu Gly Ala LeuGly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 caa ttc ctt ttaaat aca ctg tgg cca gtt aat gat aca gct ata tgg 336 Gln Phe Leu Leu AsnThr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp 100 105 110 gaa gct ttc atgcga cag gtg gag gaa ctt gtc aat caa caa ata aca 384 Glu Ala Phe Met ArgGln Val Glu Glu Leu Val Asn Gln Gln Ile Thr 115 120 125 gaa ttt gca agaaat cag gca ctt gca aga ttg caa gga tta gga gac 432 Glu Phe Ala Arg AsnGln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135 140 tct ttt aat gtatat caa cgt tcc ctt caa aat tgg ttg gct gat cga 480 Ser Phe Asn Val TyrGln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg 145 150 155 160 aat gat acagaa aat tta agt gtt gtt cgt gct caa ttt ata gct tta 528 Asn Asp Thr GluAsn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu 165 170 175 gac ctt gatttt gtt aat gct att cca ttg ttt gca gta aat gga cag 576 Asp Leu Asp PheVal Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln 180 185 190 cag gtt ccatta ctg tca gta tat gca caa gct gtg aat tta cat ttg 624 Gln Val Pro LeuLeu Ser Val Tyr Ala Gln Ala Val Asn Leu His Leu 195 200 205 tta tta ttaaaa gat gca tct ctt ttt gga gaa gga tgg gga ttc aca 672 Leu Leu Leu LysAsp Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Thr 210 215 220 cag ggg gaaatt tcc aca tat tat gac cgt caa ttg gaa cta acc gct 720 Gln Gly Glu IleSer Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Ala 225 230 235 240 aag tacact aat tac tgt gaa act tgg tat aat aca ggt tta gat cgt 768 Lys Tyr ThrAsn Tyr Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250 255 tta agagga aca aat act gaa agt tgg tta aga tat cat caa ttc cgt 816 Leu Arg GlyThr Asn Thr Glu Ser Trp Leu Arg Tyr His Gln Phe Arg 260 265 270 aga gaaatg act tta gtg gta tta gat gtt gtg gcg cta ttt cca tat 864 Arg Glu MetThr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 tat gatgta cga ctt tat cca acg gga tca aac cca cag ctt aca cgt 912 Tyr Asp ValArg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 gag gtatat aca gat ccg att gta ttt aat cca cca gct aat gtt gga 960 Glu Val TyrThr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 ctttgc cga cgt tgg ggt act aat ccc tat aat act ttt tct gag ctc 1008 Leu CysArg Arg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 gaaaat gcc ttc att cgc cca cca cat ctt ttt gat agg ctg aat agc 1056 Glu AsnAla Phe Ile Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 ttaaca atc agc agt aat cga ttt cca gtt tca tct aat ttt atg gat 1104 Leu ThrIle Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 tattgg tca gga cat acg tta cgc cgt agt tat ctg aac gat tca gca 1152 Tyr TrpSer Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 gtacaa gaa gat agt tat ggc cta att aca acc aca aga gca aca att 1200 Val GlnGlu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395 400aat ccc gga gtt gat gga aca aac cgc ata gag tca acg gca gta gat 1248 AsnPro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415ttt cgt tct gca ttg ata ggt ata tat ggc gtg aat aga gct tct ttt 1296 PheArg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe 420 425 430gtc cca gga ggc ttg ttt aat ggt acg act tct cct gct aat gga gga 1344 ValPro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435 440 445tgt aga gat ctc tat gat aca aat gat gaa tta cca cca gat gaa agt 1392 CysArg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser 450 455 460acc gga agt tca acc cat aga cta tct cat gtt acc ttt ttt agc ttt 1440 ThrGly Ser Ser Thr His Arg Leu Ser His Val Thr Phe Phe Ser Phe 465 470 475480 caa act aat cag gct gga tct ata gct aat gca gga agt gta cct act 1488Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490495 tat gtt tgg acc cgt cgt gat gtg gac ctt aat aat acg att acc cca 1536Tyr Val Trp Thr Arg Arg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505510 aat aga att aca caa tta cca ttg gta aag gca tct gca cct gtt tcg 1584Asn Arg Ile Thr Gln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520525 ggt act acg gtc tta aaa ggt cca gga ttt aca gga ggg ggt ata ctc 1632Gly Thr Thr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535540 cga aga aca act aat ggc aca ttt gga acg tta aga gta acg gtt aat 1680Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550555 560 tca cca tta aca caa caa tat cgc cta aga gtt cgt ttt gcc tca aca1728 Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565570 575 gga aat ttc agt ata agg gta ctc cgt gga ggg gtt tct atc ggt gat1776 Gly Asn Phe Ser Ile Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580585 590 gtt aga tta ggg agc aca atg aac aga ggg cag gaa cta act tac gaa1824 Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595600 605 tcc ttt ttc aca aga gag ttt act act act ggt ccg ttc aat ccg cct1872 Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610615 620 ttt aca ttt aca caa gct caa gag att cta aca gtg aat gca gaa ggt1920 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625630 635 640 gtt agc acc ggt ggt gaa tat tat ata gat aga att gaa att gtccct 1968 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro645 650 655 gtg aat ccg gca cga gaa gcg gaa gag gat tta gaa gcg gcg aagaaa 2016 Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys660 665 670 gcg 2019 Ala 8 673 PRT Artificial sequence Artificialsequence description Cry9Ca1 Glu- 164 8 Met Asn Arg Asn Asn Gln Asn GluTyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 Cys Gly Cys Pro Ser Asp AspAsp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 Pro Asn Ala Ala Leu Gln AsnMet Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 Thr Asp Glu Asp Tyr Thr AspSer Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 Ser Gly Arg Asp Ala Val GlnThr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 Leu Gly Ala Leu Gly ValPro Phe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 Gln Phe Leu Leu Asn ThrLeu Trp Pro Val Asn Asp Thr Ala Ile Trp 100 105 110 Glu Ala Phe Met ArgGln Val Glu Glu Leu Val Asn Gln Gln Ile Thr 115 120 125 Glu Phe Ala ArgAsn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135 140 Ser Phe AsnVal Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg 145 150 155 160 AsnAsp Thr Glu Asn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu 165 170 175Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln 180 185190 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn Leu His Leu 195200 205 Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Thr210 215 220 Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu Glu Leu ThrAla 225 230 235 240 Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr GlyLeu Asp Arg 245 250 255 Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg TyrHis Gln Phe Arg 260 265 270 Arg Glu Met Thr Leu Val Val Leu Asp Val ValAla Leu Phe Pro Tyr 275 280 285 Tyr Asp Val Arg Leu Tyr Pro Thr Gly SerAsn Pro Gln Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Ile Val PheAsn Pro Pro Ala Asn Val Gly 305 310 315 320 Leu Cys Arg Arg Trp Gly ThrAsn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 Glu Asn Ala Phe Ile ArgPro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 Leu Thr Ile Ser SerAsn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 Tyr Trp Ser GlyHis Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 Val Gln GluAsp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395 400 AsnPro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg Ala Ser Phe 420 425430 Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435440 445 Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser450 455 460 Thr Gly Ser Ser Thr His Arg Leu Ser His Val Thr Phe Phe SerPhe 465 470 475 480 Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala Gly SerVal Pro Thr 485 490 495 Tyr Val Trp Thr Arg Arg Asp Val Asp Leu Asn AsnThr Ile Thr Pro 500 505 510 Asn Arg Ile Thr Gln Leu Pro Leu Val Lys AlaSer Ala Pro Val Ser 515 520 525 Gly Thr Thr Val Leu Lys Gly Pro Gly PheThr Gly Gly Gly Ile Leu 530 535 540 Arg Arg Thr Thr Asn Gly Thr Phe GlyThr Leu Arg Val Thr Val Asn 545 550 555 560 Ser Pro Leu Thr Gln Gln TyrArg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 Gly Asn Phe Ser Ile ArgVal Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 Val Arg Leu Gly SerThr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 Ser Phe Phe ThrArg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615 620 Phe Thr PheThr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625 630 635 640 ValSer Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys 660 665670 Ala 9 2019 DNA Artificial sequence Artificial sequence descriptionCry9Ca1-100% 9 atg aat cga aat aat caa aat gaa tat gaa att att gaa gccccc cat 48 Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Glu Ala ProHis 1 5 10 15 tgt ggg tgt cca tca gaa gaa gaa tta agg tat cct ttg gcaagt gaa 96 Cys Gly Cys Pro Ser Glu Glu Glu Leu Arg Tyr Pro Leu Ala SerGlu 20 25 30 cca aat gca gcg tta caa aat atg aac tat aaa gaa tac tta caaatg 144 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Glu Tyr Leu Gln Met35 40 45 aca gaa gag gaa tac act gaa tct tat ata aat cct agt tta tct att192 Thr Glu Glu Glu Tyr Thr Glu Ser Tyr Ile Asn Pro Ser Leu Ser Ile 5055 60 agt ggt aga gaa gca tta cag act gcg ctt act gtt att agg aga ata240 Ser Gly Arg Glu Ala Leu Gln Thr Ala Leu Thr Val Ile Arg Arg Ile 6570 75 80 ctc ggg gct tta ggt tta ccg ttt tct gga caa ata tta agt ttt tat288 Leu Gly Ala Leu Gly Leu Pro Phe Ser Gly Gln Ile Leu Ser Phe Tyr 8590 95 caa ttc ctt tta aat aca ctg ttt cca tta aat gaa aca gct ata ttt336 Gln Phe Leu Leu Asn Thr Leu Phe Pro Leu Asn Glu Thr Ala Ile Phe 100105 110 gaa gct ttc atg cga cag tta gag gaa ctt tta aat caa caa ata aca384 Glu Ala Phe Met Arg Gln Leu Glu Glu Leu Leu Asn Gln Gln Ile Thr 115120 125 gaa ttt gca aga aat cag gca ctt gca aga ttg caa gga tta gga gaa432 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Glu 130135 140 tct ttt aat tta tat caa cgt tcc ctt caa aat ttt ttg gct gaa cga480 Ser Phe Asn Leu Tyr Gln Arg Ser Leu Gln Asn Phe Leu Ala Glu Arg 145150 155 160 aat gaa aca cga aat tta agt tta tta cgt gct caa ttt ata gcttta 528 Asn Glu Thr Arg Asn Leu Ser Leu Leu Arg Ala Gln Phe Ile Ala Leu165 170 175 gaa ctt gaa ttt tta aat gct att cca ttg ttt gca tta aat ggacag 576 Glu Leu Glu Phe Leu Asn Ala Ile Pro Leu Phe Ala Leu Asn Gly Gln180 185 190 cag tta cca tta ctg tca tta tat gca caa gct tta aat tta catttg 624 Gln Leu Pro Leu Leu Ser Leu Tyr Ala Gln Ala Leu Asn Leu His Leu195 200 205 tta tta tta aaa gaa gca tct ctt ttt gga gaa gga ttt gga ttcaca 672 Leu Leu Leu Lys Glu Ala Ser Leu Phe Gly Glu Gly Phe Gly Phe Thr210 215 220 cag ggg gaa att tcc aca tat tat gaa cgt caa ttg gaa cta accgct 720 Gln Gly Glu Ile Ser Thr Tyr Tyr Glu Arg Gln Leu Glu Leu Thr Ala225 230 235 240 aag tac act aat tac tgt gaa act ttt tat aat aca ggt ttagaa cgt 768 Lys Tyr Thr Asn Tyr Cys Glu Thr Phe Tyr Asn Thr Gly Leu GluArg 245 250 255 tta aga gga aca aat act gaa agt ttt tta aga tat cat caattc cgt 816 Leu Arg Gly Thr Asn Thr Glu Ser Phe Leu Arg Tyr His Gln PheArg 260 265 270 aga gaa atg act tta tta tta tta gaa tta tta gcg cta tttcca tat 864 Arg Glu Met Thr Leu Leu Leu Leu Glu Leu Leu Ala Leu Phe ProTyr 275 280 285 tat gaa tta cga ctt tat cca acg gga tca aac cca cag cttaca cgt 912 Tyr Glu Leu Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu ThrArg 290 295 300 gag tta tat aca gaa ccg att tta ttt aat cca cca gct aattta gga 960 Glu Leu Tyr Thr Glu Pro Ile Leu Phe Asn Pro Pro Ala Asn LeuGly 305 310 315 320 ctt tgc cga cgt ttt ggt act aat ccc tat aat act ttttct gag ctc 1008 Leu Cys Arg Arg Phe Gly Thr Asn Pro Tyr Asn Thr Phe SerGlu Leu 325 330 335 gaa aat gcc ttc att cgc cca cca cat ctt ttt gaa aggctg aat agc 1056 Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Glu Arg LeuAsn Ser 340 345 350 tta aca atc agc agt aat cga ttt cca tta tca tct aatttt atg gaa 1104 Leu Thr Ile Ser Ser Asn Arg Phe Pro Leu Ser Ser Asn PheMet Glu 355 360 365 tat ttt tca gga cat acg tta cgc cgt agt tat ctg aacgaa tca gca 1152 Tyr Phe Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn GluSer Ala 370 375 380 tta caa gaa gaa agt tat ggc cta att aca acc aca agagca aca att 1200 Leu Gln Glu Glu Ser Tyr Gly Leu Ile Thr Thr Thr Arg AlaThr Ile 385 390 395 400 aat ccc gga tta gaa gga aca aac cgc ata gag tcaacg gca tta gaa 1248 Asn Pro Gly Leu Glu Gly Thr Asn Arg Ile Glu Ser ThrAla Leu Glu 405 410 415 ttt cgt tct gca ttg ata ggt ata tat ggc tta aataga gct tct ttt 1296 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Leu Asn ArgAla Ser Phe 420 425 430 tta cca gga ggc ttg ttt aat ggt acg act tct cctgct aat gga gga 1344 Leu Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro AlaAsn Gly Gly 435 440 445 tgt aga gaa ctc tat gaa aca aat gaa gaa tta ccacca gaa gaa agt 1392 Cys Arg Glu Leu Tyr Glu Thr Asn Glu Glu Leu Pro ProGlu Glu Ser 450 455 460 acc gga agt tca acc cat aga cta tct cat tta accttt ttt agc ttt 1440 Thr Gly Ser Ser Thr His Arg Leu Ser His Leu Thr PhePhe Ser Phe 465 470 475 480 caa act aat cag gct gga tct ata gct aat gcagga agt tta cct act 1488 Gln Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala GlySer Leu Pro Thr 485 490 495 tat tta ttt acc cgt cgt gaa tta gaa ctt aataat acg att acc cca 1536 Tyr Leu Phe Thr Arg Arg Glu Leu Glu Leu Asn AsnThr Ile Thr Pro 500 505 510 aat aga att aca caa tta cca ttg tta aag gcatct gca cct tta tcg 1584 Asn Arg Ile Thr Gln Leu Pro Leu Leu Lys Ala SerAla Pro Leu Ser 515 520 525 ggt act acg tta tta aaa ggt cca gga ttt acagga ggg ggt ata ctc 1632 Gly Thr Thr Leu Leu Lys Gly Pro Gly Phe Thr GlyGly Gly Ile Leu 530 535 540 cga aga aca act aat ggc aca ttt gga acg ttaaga tta acg tta aat 1680 Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu ArgLeu Thr Leu Asn 545 550 555 560 tca cca tta aca caa caa tat cgc cta agatta cgt ttt gcc tca aca 1728 Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg LeuArg Phe Ala Ser Thr 565 570 575 gga aat ttc agt ata agg tta ctc cgt ggaggg tta tct atc ggt gaa 1776 Gly Asn Phe Ser Ile Arg Leu Leu Arg Gly GlyLeu Ser Ile Gly Glu 580 585 590 tta aga tta ggg agc aca atg aac aga gggcag gaa cta act tac gaa 1824 Leu Arg Leu Gly Ser Thr Met Asn Arg Gly GlnGlu Leu Thr Tyr Glu 595 600 605 tcc ttt ttc aca aga gag ttt act act actggt ccg ttc aat ccg cct 1872 Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr GlyPro Phe Asn Pro Pro 610 615 620 ttt aca ttt aca caa gct caa gag att ctaaca tta aat gca gaa ggt 1920 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu ThrLeu Asn Ala Glu Gly 625 630 635 640 tta agc acc ggt ggt gaa tat tat atagaa aga att gaa att tta cct 1968 Leu Ser Thr Gly Gly Glu Tyr Tyr Ile GluArg Ile Glu Ile Leu Pro 645 650 655 tta aat ccg gca cga gaa gcg gaa gaggaa tta gaa gcg gcg aag aaa 2016 Leu Asn Pro Ala Arg Glu Ala Glu Glu GluLeu Glu Ala Ala Lys Lys 660 665 670 gcg 2019 Ala 10 673 PRT Artificialsequence Artificial sequence description Cry9Ca1-100% 10 Met Asn Arg AsnAsn Gln Asn Glu Tyr Glu Ile Ile Glu Ala Pro His 1 5 10 15 Cys Gly CysPro Ser Glu Glu Glu Leu Arg Tyr Pro Leu Ala Ser Glu 20 25 30 Pro Asn AlaAla Leu Gln Asn Met Asn Tyr Lys Glu Tyr Leu Gln Met 35 40 45 Thr Glu GluGlu Tyr Thr Glu Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 Ser Gly ArgGlu Ala Leu Gln Thr Ala Leu Thr Val Ile Arg Arg Ile 65 70 75 80 Leu GlyAla Leu Gly Leu Pro Phe Ser Gly Gln Ile Leu Ser Phe Tyr 85 90 95 Gln PheLeu Leu Asn Thr Leu Phe Pro Leu Asn Glu Thr Ala Ile Phe 100 105 110 GluAla Phe Met Arg Gln Leu Glu Glu Leu Leu Asn Gln Gln Ile Thr 115 120 125Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Glu 130 135140 Ser Phe Asn Leu Tyr Gln Arg Ser Leu Gln Asn Phe Leu Ala Glu Arg 145150 155 160 Asn Glu Thr Arg Asn Leu Ser Leu Leu Arg Ala Gln Phe Ile AlaLeu 165 170 175 Glu Leu Glu Phe Leu Asn Ala Ile Pro Leu Phe Ala Leu AsnGly Gln 180 185 190 Gln Leu Pro Leu Leu Ser Leu Tyr Ala Gln Ala Leu AsnLeu His Leu 195 200 205 Leu Leu Leu Lys Glu Ala Ser Leu Phe Gly Glu GlyPhe Gly Phe Thr 210 215 220 Gln Gly Glu Ile Ser Thr Tyr Tyr Glu Arg GlnLeu Glu Leu Thr Ala 225 230 235 240 Lys Tyr Thr Asn Tyr Cys Glu Thr PheTyr Asn Thr Gly Leu Glu Arg 245 250 255 Leu Arg Gly Thr Asn Thr Glu SerPhe Leu Arg Tyr His Gln Phe Arg 260 265 270 Arg Glu Met Thr Leu Leu LeuLeu Glu Leu Leu Ala Leu Phe Pro Tyr 275 280 285 Tyr Glu Leu Arg Leu TyrPro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 Glu Leu Tyr Thr GluPro Ile Leu Phe Asn Pro Pro Ala Asn Leu Gly 305 310 315 320 Leu Cys ArgArg Phe Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 Glu AsnAla Phe Ile Arg Pro Pro His Leu Phe Glu Arg Leu Asn Ser 340 345 350 LeuThr Ile Ser Ser Asn Arg Phe Pro Leu Ser Ser Asn Phe Met Glu 355 360 365Tyr Phe Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Glu Ser Ala 370 375380 Leu Gln Glu Glu Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385390 395 400 Asn Pro Gly Leu Glu Gly Thr Asn Arg Ile Glu Ser Thr Ala LeuGlu 405 410 415 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Leu Asn Arg AlaSer Phe 420 425 430 Leu Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro AlaAsn Gly Gly 435 440 445 Cys Arg Glu Leu Tyr Glu Thr Asn Glu Glu Leu ProPro Glu Glu Ser 450 455 460 Thr Gly Ser Ser Thr His Arg Leu Ser His LeuThr Phe Phe Ser Phe 465 470 475 480 Gln Thr Asn Gln Ala Gly Ser Ile AlaAsn Ala Gly Ser Leu Pro Thr 485 490 495 Tyr Leu Phe Thr Arg Arg Glu LeuGlu Leu Asn Asn Thr Ile Thr Pro 500 505 510 Asn Arg Ile Thr Gln Leu ProLeu Leu Lys Ala Ser Ala Pro Leu Ser 515 520 525 Gly Thr Thr Leu Leu LysGly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 Arg Arg Thr Thr AsnGly Thr Phe Gly Thr Leu Arg Leu Thr Leu Asn 545 550 555 560 Ser Pro LeuThr Gln Gln Tyr Arg Leu Arg Leu Arg Phe Ala Ser Thr 565 570 575 Gly AsnPhe Ser Ile Arg Leu Leu Arg Gly Gly Leu Ser Ile Gly Glu 580 585 590 LeuArg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615620 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Leu Asn Ala Glu Gly 625630 635 640 Leu Ser Thr Gly Gly Glu Tyr Tyr Ile Glu Arg Ile Glu Ile LeuPro 645 650 655 Leu Asn Pro Ala Arg Glu Ala Glu Glu Glu Leu Glu Ala AlaLys Lys 660 665 670 Ala 11 2019 DNA Artificial sequence Artificialsequence description Cry9Ca1-25% 11 atg aat cga aat aat caa aat gaa tatgaa att att gat gcc ccc cat 48 Met Asn Arg Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala Pro His 1 5 10 15 tgt ggg tgt cca tca gat gac gat gtgagg tat cct ttg gca agt gac 96 Cys Gly Cys Pro Ser Asp Asp Asp Val ArgTyr Pro Leu Ala Ser Asp 20 25 30 cca aat gca gcg tta caa aat atg aac tataaa gat tac tta caa atg 144 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr LysAsp Tyr Leu Gln Met 35 40 45 aca gat gag gac tac act gat tct tat ata aatcct agt tta tct att 192 Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn ProSer Leu Ser Ile 50 55 60 agt ggt aga gaa gca tta cag act gcg ctt acg ttatta ggg aga ata 240 Ser Gly Arg Glu Ala Leu Gln Thr Ala Leu Thr Leu LeuGly Arg Ile 65 70 75 80 ctc ggg gct tta ggt gtt ccg ttt tct gga caa atatta agt ttt tat 288 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile LeuSer Phe Tyr 85 90 95 caa ttc ctt tta aat aca ctg tgg cca gtt aat gat acagct ata tgg 336 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr AlaIle Trp 100 105 110 gaa gct ttc atg cga cag gtg gag gaa ctt gtc aat caacaa ata aca 384 Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln GlnIle Thr 115 120 125 gaa ttt gca aga aat cag gca ctt gca aga ttg caa ggatta gga gaa 432 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly LeuGly Glu 130 135 140 tct ttt aat gta tat caa cgt tcc ctt caa aat tgg ttggct gat cga 480 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu AlaAsp Arg 145 150 155 160 aat gat aca cga aat tta agt tta tta cgt gct caattt ata gct tta 528 Asn Asp Thr Arg Asn Leu Ser Leu Leu Arg Ala Gln PheIle Ala Leu 165 170 175 gac ctt gat ttt gtt aat gct att cca ttg ttt gcagta aat gga cag 576 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala ValAsn Gly Gln 180 185 190 cag gtt cca tta ctg tca gta tat gca caa gct ttaaat tta cat ttg 624 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Leu AsnLeu His Leu 195 200 205 tta tta tta aaa gaa gca tct ctt ttt gga gaa ggatgg gga ttc aca 672 Leu Leu Leu Lys Glu Ala Ser Leu Phe Gly Glu Gly TrpGly Phe Thr 210 215 220 cag ggg gaa att tcc aca tat tat gaa cgt caa ttggaa cta acc gct 720 Gln Gly Glu Ile Ser Thr Tyr Tyr Glu Arg Gln Leu GluLeu Thr Ala 225 230 235 240 aag tac act aat tac tgt gaa act tgg tat aataca ggt tta gaa cgt 768 Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn ThrGly Leu Glu Arg 245 250 255 tta aga gga aca aat act gaa agt ttt tta agatat cat caa ttc cgt 816 Leu Arg Gly Thr Asn Thr Glu Ser Phe Leu Arg TyrHis Gln Phe Arg 260 265 270 aga gaa atg act tta gtg gta tta gat gtt gtggcg cta ttt cca tat 864 Arg Glu Met Thr Leu Val Val Leu Asp Val Val AlaLeu Phe Pro Tyr 275 280 285 tat gat gta cga ctt tat cca acg gga tca aaccca cag ctt aca cgt 912 Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn ProGln Leu Thr Arg 290 295 300 gag gta tat aca gat ccg att gta ttt aat ccacca gct aat tta gga 960 Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro ProAla Asn Leu Gly 305 310 315 320 ctt tgc cga cgt tgg ggt act aat ccc tataat act ttt tct gag ctc 1008 Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr AsnThr Phe Ser Glu Leu 325 330 335 gaa aat gcc ttc att cgc cca cca cat cttttt gaa agg ctg aat agc 1056 Glu Asn Ala Phe Ile Arg Pro Pro His Leu PheGlu Arg Leu Asn Ser 340 345 350 tta aca atc agc agt aat cga ttt cca gtttca tct aat ttt atg gaa 1104 Leu Thr Ile Ser Ser Asn Arg Phe Pro Val SerSer Asn Phe Met Glu 355 360 365 tat ttt tca gga cat acg tta cgc cgt agttat ctg aac gat tca gca 1152 Tyr Phe Ser Gly His Thr Leu Arg Arg Ser TyrLeu Asn Asp Ser Ala 370 375 380 gta caa gaa gat agt tat ggc cta att acaacc aca aga gca aca att 1200 Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr ThrThr Arg Ala Thr Ile 385 390 395 400 aat ccc gga gtt gat gga aca aac cgcata gag tca acg gca gta gat 1248 Asn Pro Gly Val Asp Gly Thr Asn Arg IleGlu Ser Thr Ala Val Asp 405 410 415 ttt cgt tct gca ttg ata ggt ata tatggc gtg aat aga gct tct ttt 1296 Phe Arg Ser Ala Leu Ile Gly Ile Tyr GlyVal Asn Arg Ala Ser Phe 420 425 430 gtc cca gga ggc ttg ttt aat ggt acgact tct cct gct aat gga gga 1344 Val Pro Gly Gly Leu Phe Asn Gly Thr ThrSer Pro Ala Asn Gly Gly 435 440 445 tgt aga gat ctc tat gat aca aat gatgaa tta cca cca gat gaa agt 1392 Cys Arg Asp Leu Tyr Asp Thr Asn Asp GluLeu Pro Pro Asp Glu Ser 450 455 460 acc gga agt tca acc cat aga cta tctcat tta acc ttt ttt agc ttt 1440 Thr Gly Ser Ser Thr His Arg Leu Ser HisLeu Thr Phe Phe Ser Phe 465 470 475 480 caa act aat cag gct gga tct atagct aat gca gga agt gta cct act 1488 Gln Thr Asn Gln Ala Gly Ser Ile AlaAsn Ala Gly Ser Val Pro Thr 485 490 495 tat gtt tgg acc cgt cgt gat gtggac ctt aat aat acg att acc cca 1536 Tyr Val Trp Thr Arg Arg Asp Val AspLeu Asn Asn Thr Ile Thr Pro 500 505 510 aat aga att aca caa tta cca ttggta aag gca tct gca cct gtt tcg 1584 Asn Arg Ile Thr Gln Leu Pro Leu ValLys Ala Ser Ala Pro Val Ser 515 520 525 ggt act acg gtc tta aaa ggt ccagga ttt aca gga ggg ggt ata ctc 1632 Gly Thr Thr Val Leu Lys Gly Pro GlyPhe Thr Gly Gly Gly Ile Leu 530 535 540 cga aga aca act aat ggc aca tttgga acg tta aga gta acg gtt aat 1680 Arg Arg Thr Thr Asn Gly Thr Phe GlyThr Leu Arg Val Thr Val Asn 545 550 555 560 tca cca tta aca caa caa tatcgc cta aga tta cgt ttt gcc tca aca 1728 Ser Pro Leu Thr Gln Gln Tyr ArgLeu Arg Leu Arg Phe Ala Ser Thr 565 570 575 gga aat ttc agt ata agg gtactc cgt gga ggg gtt tct atc ggt gat 1776 Gly Asn Phe Ser Ile Arg Val LeuArg Gly Gly Val Ser Ile Gly Asp 580 585 590 gtt aga tta ggg agc aca atgaac aga ggg cag gaa cta act tac gaa 1824 Val Arg Leu Gly Ser Thr Met AsnArg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 tcc ttt ttc aca aga gag tttact act act ggt ccg ttc aat ccg cct 1872 Ser Phe Phe Thr Arg Glu Phe ThrThr Thr Gly Pro Phe Asn Pro Pro 610 615 620 ttt aca ttt aca caa gct caagag att cta aca gtg aat gca gaa ggt 1920 Phe Thr Phe Thr Gln Ala Gln GluIle Leu Thr Val Asn Ala Glu Gly 625 630 635 640 gtt agc acc ggt ggt gaatat tat ata gat aga att gaa att gtc cct 1968 Val Ser Thr Gly Gly Glu TyrTyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655 gtg aat ccg gca cga gaagcg gaa gag gat tta gaa gcg gcg aag aaa 2016 Val Asn Pro Ala Arg Glu AlaGlu Glu Asp Leu Glu Ala Ala Lys Lys 660 665 670 gcg 2019 Ala 12 673 PRTArtificial sequence Artificial sequence description Cry9Ca1-25% 12 MetAsn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60Ser Gly Arg Glu Ala Leu Gln Thr Ala Leu Thr Leu Leu Gly Arg Ile 65 70 7580 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Leu Ser Phe Tyr 85 9095 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp 100105 110 Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln Ile Thr115 120 125 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu GlyGlu 130 135 140 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu AlaAsp Arg 145 150 155 160 Asn Asp Thr Arg Asn Leu Ser Leu Leu Arg Ala GlnPhe Ile Ala Leu 165 170 175 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu PheAla Val Asn Gly Gln 180 185 190 Gln Val Pro Leu Leu Ser Val Tyr Ala GlnAla Leu Asn Leu His Leu 195 200 205 Leu Leu Leu Lys Glu Ala Ser Leu PheGly Glu Gly Trp Gly Phe Thr 210 215 220 Gln Gly Glu Ile Ser Thr Tyr TyrGlu Arg Gln Leu Glu Leu Thr Ala 225 230 235 240 Lys Tyr Thr Asn Tyr CysGlu Thr Trp Tyr Asn Thr Gly Leu Glu Arg 245 250 255 Leu Arg Gly Thr AsnThr Glu Ser Phe Leu Arg Tyr His Gln Phe Arg 260 265 270 Arg Glu Met ThrLeu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 Tyr Asp ValArg Leu Tyr Pro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 Glu ValTyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Leu Gly 305 310 315 320Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330335 Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Glu Arg Leu Asn Ser 340345 350 Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Glu355 360 365 Tyr Phe Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp SerAla 370 375 380 Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg AlaThr Ile 385 390 395 400 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Glu SerThr Ala Val Asp 405 410 415 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly ValAsn Arg Ala Ser Phe 420 425 430 Val Pro Gly Gly Leu Phe Asn Gly Thr ThrSer Pro Ala Asn Gly Gly 435 440 445 Cys Arg Asp Leu Tyr Asp Thr Asn AspGlu Leu Pro Pro Asp Glu Ser 450 455 460 Thr Gly Ser Ser Thr His Arg LeuSer His Leu Thr Phe Phe Ser Phe 465 470 475 480 Gln Thr Asn Gln Ala GlySer Ile Ala Asn Ala Gly Ser Val Pro Thr 485 490 495 Tyr Val Trp Thr ArgArg Asp Val Asp Leu Asn Asn Thr Ile Thr Pro 500 505 510 Asn Arg Ile ThrGln Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 Gly Thr ThrVal Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 Arg ArgThr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560Ser Pro Leu Thr Gln Gln Tyr Arg Leu Arg Leu Arg Phe Ala Ser Thr 565 570575 Gly Asn Phe Ser Ile Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580585 590 Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu595 600 605 Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn ProPro 610 615 620 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn AlaGlu Gly 625 630 635 640 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg IleGlu Ile Val Pro 645 650 655 Val Asn Pro Ala Arg Glu Ala Glu Glu Asp LeuGlu Ala Ala Lys Lys 660 665 670 Ala 13 30 DNA Artificial sequenceArtificial sequence description mutant 1 13 gaattaaatg aatttttaaatttaagtgtt 30 14 30 DNA Artificial sequence Artificial sequencedescription mutant 2 14 gaattaaatg aattattaaa tttaagtgtt 30 15 30 DNAArtificial sequence Artificial sequence description mutant 3 15gaattattag aatttttatt attaagtgtt 30 16 30 DNA Artificial sequenceArtificial sequence description mutant 4 16 gaattattag aattattattattaagtgtt 30 17 30 DNA Artificial sequence Artificial sequencedescription mutant 5 17 gaattattag aagaattatt attaagtgtt 30 18 30 DNAArtificial sequence Artificial sequence description mutant 6 18gaacgattag aatttttatt attaagtgtt 30 19 30 DNA Artificial sequenceArtificial sequence description mutant 7 19 gaacgattag aattattattattaagtgtt 30 20 30 DNA Artificial sequence Artificial sequencedescription mutant 8 20 gaattagaag aattattatt attaagtgtt 30 21 30 DNAArtificial sequence Artificial sequence description mutant 9 21gaattattag aagaagaaga attaagtgtt 30 22 33 DNA Artificial sequenceArtificial sequence description mutant 10 22 tttttattaa atttattttttttaccatta ctg 33 23 33 DNA Artificial sequence Artificial sequencedescription mutant11 23 tttttattaa atttagaaga attaccatta ctg 33 24 33DNA Artificial sequence Artificial sequence description mutant 12 24tttgaagaaa atttagaaga attaccatta ctg 33 25 33 DNA Artificial sequenceArtificial sequence description mutant 13 25 tttgaagaaa attttttattatttccatta ctg 33 26 33 DNA Artificial sequence Artificial sequencedescription mutant 14 26 tttgaagaaa attttgaaga atttccatta ctg 33 27 33DNA Artificial sequence Artificial sequence description mutant 15 27tttttattaa attttgaaga atttccatta ctg 33 28 33 DNA Artificial sequenceArtificial sequence description mutant 16 28 tttttattaa atgaattttttgaaccatta ctg 33 29 24 DNA Artificial sequence Artificial sequencedescription mutant 17 29 ctttttttag aattattttt attc 24 30 24 DNAArtificial sequence Artificial sequence description mutant 18 30ctttttttat tattattttt attc 24 31 24 DNA Artificial sequence Artificialsequence description mutant 19 31 ctttttttag aagaatttga atta 24 32 24DNA Artificial sequence Artificial sequence description mutant 20 32ctttttgaag aagaatttga atta 24 33 24 DNA Artificial sequence Artificialsequence description mutant 21 33 ctttttgaag aattatttga agaa 24 34 15DNA Artificial sequence Artificial sequence description mutant 22 34ttattagaat taaat 15 35 15 DNA Artificial sequence Artificial sequencedescription mutant 23 35 ttattatttt taaat 15 36 15 DNA Artificialsequence Artificial sequence description mutant 24 36 ttagaattat taaat15 37 15 DNA Artificial sequence Artificial sequence description mutant25 37 ttattatttt ttaat 15 38 15 DNA Artificial sequence Artificialsequence description mutant 26 38 ttagaagaat taaat 15 39 15 DNAArtificial sequence Artificial sequence description mutant 27 39ttagaatttt taaat 15 40 15 DNA Artificial sequence Artificial sequencedescription mutant 28 40 ttagaatttg aaaat 15 41 15 DNA Artificialsequence Artificial sequence description mutant 29 41 ttagaagaag aaaat15 42 33 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 1 42 gatcgaaatg atacattaaa tttaagtgtt gtt 33 43 33 DNAArtificial sequence Artificial sequence description oligonucleotide 2 43gatcgaaatg atacatttaa tttaagtgtt gtt 33 44 33 DNA Artificial sequenceArtificial sequence description oligonucleotide 3 44 gatcgaaatgatacagaaaa tttaagtgtt gtt 33 45 33 DNA Artificial sequence Artificialsequence description oligonucleotide 4 45 cgaaatgata cacgattattaagtgttgtt cgt 33 46 33 DNA Artificial sequence Artificial sequencedescription oligonucleotide 5 46 cgaaatgata cacgagaatt aagtgttgtt cgt 3347 39 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 6 47 ttggctgatc gaaatgaatt tttaaattta agtgttgtt 39 48 39DNA Artificial sequence Artificial sequence description oligonucleotide7 48 ttggctgatc gaaatgaatt tttattatta agtgttgtt 39 49 39 DNA Artificialsequence Artificial sequence description oligonucleotide 8 49 ttggctgatcgaaatgaatt attaaattta agtgttgtt 39 50 39 DNA Artificial sequenceArtificial sequence description oligonucleotide 9 50 ttggctgatcgaaatgaatt attattatta agtgttgtt 39 51 39 DNA Artificial sequenceArtificial sequence description oligonucleotide 10 51 ttggctgatcgaaatgaaga agaagaatta agtgttgtt 39 52 39 DNA Artificial sequenceArtificial sequence description oligonucleotide 11 52 ttggctgatcgaaatgaaga attattatta agtgttgtt 39 53 36 DNA Artificial sequenceArtificial sequence description oligonucleotide 12 53 caaaattggttggctgaatt aaatgaatta ttaaat 36 54 36 DNA Artificial sequence Artificialsequence description oligonucleotide 13 54 caaaattggt tggctgaattaaatgaattt ttaaat 36 55 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 14 55 caaaattggt tggctgaatt attagaatttttattatta 39 56 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 15 56 caaaattggt tggctgaatt attagaattattattatta 39 57 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 16 57 caaaattggt tggctgaatt attagaagaattattatta 39 58 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 17 58 caaaattggt tggctgaacg attagaatttttattatta 39 59 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 18 59 caaaattggt tggctgaacg attagaattattattatta 39 60 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 19 60 caaaattggt tggctgaatt agaagaattattattatta 39 61 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 20 61 caaaattggt tggctgaatt attagaagaagaagaatta 39 62 36 DNA Artificial sequence Artificial sequencedescription oligonucleotide 21 62 gctattccat tgtttttatt aaatggacagcaggtt 36 63 36 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 22 63 gctattccat tgtttgaaga aaatggacag caggtt 36 64 36DNA Artificial sequence Artificial sequence description oligonucleotide23 64 ttattaaatg gacagcagtt accattactg tcagta 36 65 36 DNA Artificialsequence Artificial sequence description oligonucleotide 24 65ttattaaatg gacagcagtt tccattactg tcagta 36 66 36 DNA Artificial sequenceArtificial sequence description oligonucleotide 25 66 ttattaaatggacagcagga accattactg tcagta 36 67 36 DNA Artificial sequence Artificialsequence description oligonucleotide 26 67 gaagaaaatg gacagcagttaccattactg tcagta 36 68 36 DNA Artificial sequence Artificial sequencedescription oligonucleotide 27 68 gaagaaaatg gacagcagtt tccattactgtcagta 36 69 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 28 69 ccattgtttt tattaaattt atttttttta ccattactgt cagta45 70 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 29 70 ccattgtttt tattaaattt agaagaatta ccattactgt cagta45 71 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 30 71 ccattgtttg aagaaaattt agaagaatta ccattactgt cagta45 72 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 31 72 ccattgtttg aagaaaattt tttattattt ccattactgt cagta45 73 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 32 73 ccattgtttg aagaaaattt tgaagaattt ccattactgt cagta45 74 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 33 74 ccattgtttt tattaaattt tgaagaattt ccattactgt cagta45 75 45 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 34 75 ccattgtttt tattaaatga attttttgaa ccattactgt cagta45 76 33 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 35 76 gatgcatctc tttttttaga aggatgggga ttc 33 77 36 DNAArtificial sequence Artificial sequence description oligonucleotide 3677 gatgcatctc tttttttatt aggatgggga ttcaca 36 78 33 DNA Artificialsequence Artificial sequence description oligonucleotide 37 78gatgcatctc tttttgaaga aggatgggga ttc 33 79 33 DNA Artificial sequenceArtificial sequence description oligonucleotide 38 79 ttagaaggatggggattaac acagggggaa att 33 80 33 DNA Artificial sequence Artificialsequence description oligonucleotide 39 80 gaagaaggat ggggagaaacacagggggaa att 33 81 45 DNA Artificial sequence Artificial sequencedescription oligonucleotide 40 81 gcatctcttt ttttagaatt atttttattcacacaggggg aaatt 45 82 45 DNA Artificial sequence Artificial sequencedescription oligonucleotide 41 82 gcatctcttt ttttattatt atttttattcacacaggggg aaatt 45 83 45 DNA Artificial sequence Artificial sequencedescription oligonucleotide 42 83 gcatctcttt ttttagaatt atttttattcacacaggggg aaatt 45 84 45 DNA Artificial sequence Artificial sequencedescription oligonucleotide 43 84 gcatctcttt ttgaagaatt atttttattcacacaggggg aaatt 45 85 45 DNA Artificial sequence Artificial sequencedescription oligonucleotide 44 85 gcatctcttt ttgaagaatt atttttagaaacacaggggg aaatt 45 86 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 45 86 ggtttagatc gtttattaga attaaatactgaaagttgg 39 87 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 46 87 ggtttagatc gtttattatt tttaaatactgaaagttgg 39 88 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 47 88 ggtttagatc gtttagaatt attaaatactgaaagttgg 39 89 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 48 89 ggtttagatc gtttattatt ttttaatactgaaagttgg 39 90 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 49 90 ggtttagatc gtttagaaga attaaatactgaaagttgg 39 91 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 50 91 ggtttagatc gtttagaatt tttaaatactgaaagttgg 39 92 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 51 92 ggtttagatc gtttagaatt tgaaaatactgaaagttgg 39 93 39 DNA Artificial sequence Artificial sequencedescription oligonucleotide 52 93 ggtttagatc gtttagaaga agaaaatactgaaagttgg 39 94 30 DNA Artificial sequence Artificial sequencedescription oligonucleotide 53 94 tgaatatgaa attattgaag ccccccattg 30 9540 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 54 95 tgggtgtcca tcagaagaag aattaaggta tcctttggca 40 9627 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 55 96 tcctttggca agtgaaccaa atgcagc 27 97 25 DNAArtificial sequence Artificial sequence description oligonucleotide 5697 gaactataaa gaatacttac aaatg 25 98 26 DNA Artificial sequenceArtificial sequence description oligonucleotide 57 98 caaatgacagaagaggaata cactga 26 99 20 DNA Artificial sequence Artificial sequencedescription oligonucleotide 58 99 tacactgaat cttatataaa 20 100 36 DNAArtificial sequence Artificial sequence description oligonucleotide 59100 tattagtggt agagaagcat tacagactgc gcttac 36 101 37 DNA Artificialsequence Artificial sequence description oligonucleotide 60 101cagactgcgc ttactgttat tagggagaat actcggg 37 102 25 DNA Artificialsequence Artificial sequence description oligonucleotide 61 102gggctttagg tttaccgttt tctgg 25 103 28 DNA Artificial sequence Artificialsequence description oligonucleotide 62 103 ttctggacaa atattaagtttttatcaa 28 104 40 DNA Artificial sequence Artificial sequencedescription oligonucleotide 63 104 cttttaaata cactgtttcc attaaatgaaacagctatat 40 105 24 DNA Artificial sequence Artificial sequencedescription oligonucleotide 64 105 acagctatat ttgaagcttt catg 24 106 26DNA Artificial sequence Artificial sequence description oligonucleotide65 106 ctttcatgcg acagttagag gaactt 26 107 26 DNA Artificial sequenceArtificial sequence description oligonucleotide 66 107 gaggaacttttaaatcaaca aataac 26 108 21 DNA Artificial sequence Artificial sequencedescription oligonucleotide 67 108 ggattaggag aatcttttaa t 21 109 23 DNAArtificial sequence Artificial sequence description oligonucleotide 68109 tcttttaatt tatatcaacg ttc 23 110 21 DNA Artificial sequenceArtificial sequence description oligonucleotide 69 110 ccttcaaaattttttggctg a 21 111 17 DNA Artificial sequence Artificial sequencedescription oligonucleotide 70 111 ttggctgaac gaaatga 17 112 23 DNAArtificial sequence Artificial sequence description oligonucleotide 71112 cgaaatgaaa cacgaaattt aag 23 113 37 DNA Artificial sequenceArtificial sequence description oligonucleotide 72 113 acacgaaatttaagtttatt acgtgctcaa tttatag 37 114 48 DNA Artificial sequenceArtificial sequence description oligonucleotide 73 114 gctcaatttatagctttaga acttgaattt ttaaatgcta ttccattg 48 115 27 DNA Artificialsequence Artificial sequence description oligonucleotide 74 115ccattgtttg cattaaatgg acagcag 27 116 27 DNA Artificial sequenceArtificial sequence description oligonucleotide 75 116 ccattgtttgcattaaatgg acagcag 27 117 27 DNA Artificial sequence Artificial sequencedescription oligonucleotide 76 117 ccattactgt cattatatgc acaagct 27 11827 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 77 118 tatgcacaag ctttaaattt acatttg 27 119 23 DNAArtificial sequence Artificial sequence description oligonucleotide 78119 ttattaaaag aagcatctct ttt 23 120 25 DNA Artificial sequenceArtificial sequence description oligonucleotide 79 120 tggagaaggatttggattca cacag 25 121 24 DNA Artificial sequence Artificial sequencedescription oligonucleotide 80 121 cacatattat gaacgtcaat tgga 24 122 28DNA Artificial sequence Artificial sequence description oligonucleotide81 122 tactgtgaaa ctttttataa tacaggtt 28 123 25 DNA Artificial sequenceArtificial sequence description oligonucleotide 82 123 tacaggtttagaacgtttaa gagga 25 124 28 DNA Artificial sequence Artificial sequencedescription oligonucleotide 83 124 aatactgaaa gttttttaag atatcatc 28 12551 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 84 125 gtagagaaat gactttatta ttattagaat tattagcgctatttccatat t 51 126 27 DNA Artificial sequence Artificial sequencedescription oligonucleotide 85 126 atattatgaa ttacgacttt atccaac 27 12723 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 86 127 cttacacgtg agttatatac aga 23 128 29 DNAArtificial sequence Artificial sequence description oligonucleotide 87128 tatacagaac cgattttatt taatccacc 29 129 28 DNA Artificial sequenceArtificial sequence description oligonucleotide 88 129 ccaccagctaatttaggact ttgccgac 28 130 27 DNA Artificial sequence Artificialsequence description oligonucleotide 89 130 ctttgccgac gttttggtactaatccc 27 131 23 DNA Artificial sequence Artificial sequencedescription oligonucleotide 90 131 catctttttg aaaggctgaa tag 23 132 30DNA Artificial sequence Artificial sequence description oligonucleotide91 132 taatcgattt ccattatcat ctaattttat 30 133 36 DNA Artificialsequence Artificial sequence description oligonucleotide 92 133ctaattttat ggaatatttt tcaggacata cgttac 36 134 33 DNA Artificialsequence Artificial sequence description oligonucleotide 93 134tagttatctg aacgaatcag cattacaaga aga 33 135 20 DNA Artificial sequenceArtificial sequence description oligonucleotide 94 135 caagaagaaagttatggcct 20 136 35 DNA Artificial sequence Artificial sequencedescription oligonucleotide 95 136 caattaatcc cggattagaa ggaacaaaccgcata 35 137 30 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 96 137 gagtcaacgg cattagaatt tcgttctgca 30 138 26 DNAArtificial sequence Artificial sequence description oligonucleotide 97138 ggtatatatg gcttaaatag agcttc 26 139 30 DNA Artificial sequenceArtificial sequence description oligonucleotide 98 139 tagagcttcttttttaccag gaggcttgtt 30 140 31 DNA Artificial sequence Artificialsequence description oligonucleotide 99 140 ctgctaatgg aggatgtagagaactctatg a 31 141 17 DNA Artificial sequence Artificial sequencedescription oligonucleotide 100 141 ctctatgaaa caaatga 17 142 20 DNAArtificial sequence Artificial sequence description oligonucleotide 101142 acaaatgaag aattaccacc 20 143 27 DNA Artificial sequence Artificialsequence description oligonucleotide 102 143 attaccacca gaagaaagtaccggaag 27 144 30 DNA Artificial sequence Artificial sequencedescription oligonucleotide 103 144 agactatctc atttaacctt ttttagcttt 30145 27 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 104 145 gctaatgcag gaagtttacc tacttat 27 146 26 DNAArtificial sequence Artificial sequence description oligonucleotide 105146 cctacttatt tatttacccg tcgtga 26 147 33 DNA Artificial sequenceArtificial sequence description oligonucleotide 106 147 acccgtcgtgaattagaact taataatacg att 33 148 24 DNA Artificial sequence Artificialsequence description oligonucleotide 107 148 attaccattg ttaaaggcat ctgc24 149 30 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 108 149 aaggcatctg cacctttatc gggtactacg 30 150 29 DNAArtificial sequence Artificial sequence description oligonucleotide 109150 tcgggtacta cgttattaaa aggtccagg 29 151 40 DNA Artificial sequenceArtificial sequence description oligonucleotide 110 151 acatttggaacgttaagatt aacgttaaat tcaccattaa 40 152 37 DNA Artificial sequenceArtificial sequence description oligonucleotide 111 152 cacaacaatatcgcctaaga ttacgttttg cctcaac 37 153 31 DNA Artificial sequenceArtificial sequence description oligonucleotide 112 153 aaatttcagtataaggttac tccgtggagg g 31 154 35 DNA Artificial sequence Artificialsequence description oligonucleotide 113 154 ataagggtac tccgtggagggttatctatc ggtga 35 155 29 DNA Artificial sequence Artificial sequencedescription oligonucleotide 114 155 tctatcggtg aattaagatt agggagcac 29156 30 DNA Artificial sequence Artificial sequence descriptionoligonucleotide 115 156 caagagattc taacattaaa tgcagaaggt 30 157 32 DNAArtificial sequence Artificial sequence description oligonucleotide 116157 aatgcagaag gtttaagcac cggtggtgaa ta 32 158 32 DNA Artificialsequence Artificial sequence description oligonucleotide 117 158gtggtgaata ttatatagaa agaattgaaa tt 32 159 37 DNA Artificial sequenceArtificial sequence description oligonucleotide 118 159 agaattgaaattttaccttt aaatccggca cgagaag 37 160 30 DNA Artificial sequenceArtificial sequence description oligonucleotide 119 160 cgagaagcggaagaggaatt agaagcggcg 30

1. A pepsin-sensitive modified Cry protein, characterized in that it has at least one additional pepsin cleavage site.
 2. The modified Cry protein as claimed in claim 1, characterized in that the additional pepsin cleavage site is represented by an amino acid residue chosen from leucine, phenylalanine and glutamic acid residues.
 3. The modified Cry protein as claimed in either of claims 1 and 2, characterized in that it is selected from the Cry1, Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20 proteins.
 4. The modified Cry protein as claimed in claim 3, characterized in that it is a Cry9C protein.
 5. The modified Cry protein as claimed in claim 4, characterized in that it is the Cry9Ca1 protein.
 6. The modified Cry protein as claimed in one of claims 1 to 5, characterized in that it has at least one additional pepsin cleavage site in at least one of the inter-α-helix loops of domain I.
 7. The modified Cry protein as claimed in one of claims 1 to 6, characterized in that it has at least one additional pepsin cleavage site in the inter-α-helix loop linking the α3 and α4 helices of domain I.
 8. The modified Cry protein as claimed in one of claims 5 to 7, characterized in that it has an additional pepsin cleavage site at position
 164. 9. The modified Cry protein as claimed in claim 8, characterized in that it is selected from the Cry proteins, the sequences of which are represented by the identifiers SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
 10. The modified Cry protein as claimed in one of claims 1 to 5, characterized in that the additional pepsin cleavage sites are introduced by substituting aspartic acid residues with glutamic acid residues, substituting tryptophan residues with phenylalanine residues, and substituting valine or isoleucine residues with leucine residues.
 11. The modified Cry protein as claimed in claim 11, characterized in that the degree of substitutions which said Cry protein possesses is 25%.
 12. A method for increasing the pepsin sensitivity of the Cry proteins, characterized in that at least one additional pepsin or cleavage site is introduced into said Cry proteins.
 13. The method as claimed in claim 12, characterized in that the additional pepsin cleavage site introduced is represented by an amino acid chosen from leucine, phenylalanine and glutamic acid residues.
 14. The method as claimed in either of claims 12 and 13, characterized in that it applies to the Cry proteins selected from the Cry1, Cry3, Cry4, Cry7, Cry8, Cry9, Cry10, Cry16, Cry17, Cry19 and Cry20 proteins.
 15. The method as claimed in claim 14, characterized in that it applies to the Cry9C protein.
 16. The method as claimed in claim 15, characterized in that it applies to the Cry9Ca1 protein.
 17. The method as claimed in one of claims 12 to 16, characterized in that at least one additional pepsin cleavage site is introduced into at least one of the inter-α-helix loops of domain I of said Cry proteins.
 18. The method as claimed in one of claims 12 to 17, characterized in that at least one additional pepsin cleavage site is introduced into the inter-α-helix loop linking the α and α4 helices of domain I.
 19. The method as claimed in one of claims 16 to 18, characterized in that an additional pepsin cleavage site is introduced at position
 164. 20. The method as claimed in one of claims 12 to 16, characterized in that the additional pepsin cleavage sites are introduced by substituting aspartic acid residues with glutamic acid, substituting tryptophan residues with phenylalanine residues, and substituting valine or isoleucine residues with leucine residues.
 21. The method as claimed in claim 20, characterized in that the degree of substitution which said Cry protein possesses is less than or equal to 25%.
 22. A polynucleotide encoding a modified Cry protein as claimed in one of claims 1 to
 11. 23. A chimeric gene comprising, functionally linked to one another, at least: (a) one promoter which is functional in a host organism (b) a polynucleotide as claimed in claim 22 (c) a terminator element which is functional in a host organism.
 24. The chimeric gene as claimed in claim 23, characterized in that the promoter and the terminator element are functional inplants.
 25. An expression or transformation vector containing a chimeric gene as claimed in either of claims 23 and
 24. 26. The vector as claimed in claim 27, characterized in that it is a plasmid, a phase or a virus.
 27. A host organism transformed with one of the vectors as claimed in either of claims 25 and
 26. 28. The host organism as claimed in claim 27, characterized in that it is a plant.
 29. The plant as claimed in claim 28, characterized in that it contains, in addition to a chimeric gene as claimed in either of claims 23 and 24, at least one other chimeric gene containing a polynucleotide encoding a protein of interest.
 30. A part of a plant as claimed in claim
 29. 31. A seed from a plant as claimed in claim
 29. 32. A method for producing the modified Cry proteins as claimed in one of claims 1 to 11, characterized in that it comprises at least the steps of: (a) culturing a transformed host organism according to the invention in a culture medium suitable for the growth and for the multiplication of said organism, (b) (b) extracting the Cry proteins produced by the transformed organism cultured in step (a).
 33. The method as claimed in claim 32, characterized in that it comprises a step (c) of purification of the Cry proteins extracted in step (b).
 34. The method as claimed in either of claims 32 and 33, characterized in that the host organism is a microorganism.
 35. The method as claimed in claim 34, characterized in that the host organism is a Bacillus thuringiensis bacterium.
 36. A monoclonal or polyclonal antibody, characterized in that it is directed against a modified Cry protein as claimed in one of claims 1 to
 11. 